key: cord-0008864-43zrew96
authors: Carrasco, Luis
title: Picornavirus inhibitors
date: 2002-11-25
journal: Pharmacol Ther
DOI: 10.1016/0163-7258(94)90040-x
sha: 0f0e3a135b32ddbc410dd175a7d6c559de75f9d4
doc_id: 8864
cord_uid: 43zrew96

Picornaviruses are among the best understood animal viruses in molecular terms. A number of important human and animal pathogens are members of the Picornaviridae family. The genome organization, the different steps of picornavirus growth and numerous compounds that have been reported as inhibitors of picornavirus functions are reviewed. The picornavirus particles and several agents that interact with them have been solved at atomic resolution, leading to computer-assisted drug design. Picornavirus inhibitors are useful in aiding a better understanding of picornavirus biology. In addition, some of them are promising therapeutic agents. Clinical efficacy of agents that bind to picornavirus particles has already been demonstrated.

Abbreviations--2'-5' A, ppp(A2'p5'A)nA; BFA, brefeldin A; BFLA1, bafilomycin AI; dsRNA, doublestranded RNA; EMC, encephalomyocarditis; FMDV, foot-and-mouth disease virus; (3413, 2-amino-5-(2-sulfamoylphenyl)-l,3,4-thiadiazole; HBB, 2-(ct-hydroxybenzyl)-benzimidazole; HIV, human immunodeficiency virus; HPA-23, ammonium 5-tungsto-2-antimonate; ICAM-I, intercellular adhesion molecule-l; IP3, inositol triphosphate; M12325, 5-aminosulfonyl-2,4-dichorobenzoate; 3-MQ, 3-methyl quereetin; IRES, internal ribosome entry site; L protein, leader protein; RF, replicative form; RI, replicative intermediate; RLP, ribosome landing pad; SFV, Semliki forest virus; TOFA, 5-(tetradecyloxy)-2-furoic acid; VPg, viral protein bound to the genome; VSV, vesicular stomatitis virus. 233  233  233  235  235  236  238  239  239  239  240  241  241  242  244  248  248  249  249  251  252  253  253  254  255  256  256  257  257  257  258  259  259  260  261  261  262  263  263  264  264  264  265  265  265  266  266  267  267  268  268  268  268  268  268  268  269  270  270  270  270  270  271  271  271 1. INTRODUCTION

The Picornaviridae family has been the object of interest of many research groups for more than 30 years. As a result, members of this virus family are among the best understood animal viruses in molecular terms. A number of excellent reviews have been published covering different aspects of picornavirology (Putnak and Phillips, 1981; Koch and Koch, 1985; Carrasco and Castrillo, 1987; Rossmann and Johnson, 1989; Sonenberg, 1990; Palmenberg, 1990; Stanway, 1990; Racaniello, 1990; Lawson and Semler, 1990; Richards and Ehrenfeld, 1990; Minor, 1990; Sarnow et al., 1990; Minor, 1992; Kirkegaard, 1992; Agol, 1993; Harber and Wimmer, 1993) . Picornaviruses are structurally simple and appear as spherical particles of -,~ 30 nm diameter that possess icosahedral symmetry. The genome is a molecule of single-stranded RNA of positive polarity. This family was first divided into four genera: Enterovirus, Cardiovirus, Rhinovirus and Aphtovirus. More recently, an additional genus, Hepatovirus, has been recognized on the basis of genome sequence and organization (Fig. 1) . Some members of this group are of great importance in medicine and veterinary science, because they are pathogens of humans or of animals and, hence, of economic or ecological interest. Examples include poliovirus, the causative agent of poliomyelitis, coxsackie virus, a factor responsible for causing heart disease, and echo viruses that cause meningitis in humans, all three belonging to the Enterovirus genus. The main causative agents of common colds are rhinoviruses (genus Rhinovirus). Hepatitis A, a virus that causes hepatitis, was previously classified in the Enterovirus genus, but is now included in the Hepatovirus genus (Stanway, 1990; Harber and Wimmer, 1993) . Finally, the infective agent for one of the most threatening afflictions of cattle, foot-and-mouth disease, is a member of the Aphtovirus genus. Although effective vaccines have been developed for some of these viruses, such as poliovirus or hepatitis A virus, the development of vaccines for other members of this group, such as rhinoviruses, has yet to be achieved Murdin et al., 1990; Minor, 1990 Minor, , 1992 . Thus, it is important to develop antiviral agents to combat disease. Apart from the use of antiviral agents in this respect, these compounds play a pivotal role in molecular virology. Indeed, antiviral agents are very useful for elucidating numerous viral processes, if we can identify the exact target of the agent at the molecular level. In addition, antiviral agents provide clean and accurate tools that aid in the dissection of the complicated molecular steps of viral genome replication. Hence, antiviral agents are to molecular virology what other inhibitors are to molecular biology. Picornavirus inhibitors represent good examples of compounds that are not only useful in providing a better understanding of the biology of picornaviruses, but also in furnishing promising agents that are being assayed in clinical trials against the common cold, for instance. The large number of compounds now available for the selective blockade of different steps of the picornavirus replication cycle makes these agents invaluable tools for picornavirologists, but it is difficult to organize a comprehensive review covering all of them. Although I have tried to include as many examples as possible of the approaches used and the agents employed, inevitably, coverage will be incomplete. I apologise in advance to those whose work is not included, despite its merits.

I shall not review here the structure of the picornavirus particle, or the different steps of its replication cycle; these topics will be covered at the beginning of each section dealing with specific picornavirus inhibitors. I shall briefly outline, instead, the structure of the genome by taking poliovirus as a representative member of this viral family.

Poliovirus was the first picornavirus and, in fact, the first animal virus with an RNA genome to be sequenced (Kitamura et al., 1981; Racaniello and Baltimore, 1981a) . Soon after this achievement, infectious eDNA was obtained, thus opening new possibilities for the genetic manipulation of this important human pathogen (Racaniello and Baltimore, 1981b; Omata et al., 1984; Sarnow et al., 1990; Kirkegaard, 1992) . The RNA genome is a 7.544 stretch of nucleotides, of which almost 90% encodes for proteins (Sarnow et al., 1990; Harber and Wimmer, 1993) (Fig. 2) . The AUG that initiates protein synthesis is located at position 754 and is preceded by several AUGs (6-8, depending on the virus serotype), which are not recognized by the translation initiation complex . The initiator AUG is followed by 2209 codons that code for a polyprotein that is cleaved by virus-encoded proteases Lawson and Semler, 1990; Palmenberg, 1990) . Two termination codons are followed by 65 nucleotides, which precede a poly(A) stretch with an average length of 75 nucleotides (Yogo and Wimmer, 1972) . This is genetically determined and encoded by the genome. After infection, the viral RNA contains longer poly(A) tails of 150-200 nucleotides that, presumably, are added after replication (Spector and Baltimore, 1975a, b; Spector et ai., 1975) . The length of the genome and the disposition of the different viral proteins varies somewhat among the different picornavirus genera, as depicted in Fig. 1 . Perhaps the most remarkable variations are the presence of an additional L (leader) protein (Strebel and Beck, 1986) in foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus; the coding for three copies of the protein 3B (VPg), the virtual lack of protein 2A (16 amino acids) in FMDV (Stanway, 1990) and the presence ofa poly(C) tract of variable length in the 5" leader region in ,4phtovirus and Cardiovirus (Clarke et al., 1987; Stanway, 1990 ).

The 754 nucleotides located at the 5'-terminus of the poliovirus genome have a number of functions, of which some have yet to be fully elucidated (Sarnow et al., 1990; Kirkegaard, 1992) . Among these, the following may be listed: (1) This region is necessary for replication of the RNA genome.

(2) At least part of the 754 nucleotides is required to initiate the synthesis of the polyprotein. (3) This region is involved in genome packaging into virion particles. (4) Poliovirus neurovirulence mutants map in this region. In addition, the 5' leader sequence may be involved in other processes required for virus replication. Thus, one of the viral proteases, protein 3C or its precursor 3CD, can interact with this region and, perhaps, regulate genome replication (Andino et al., 1990a, b) . Furthermore, a poliovirus-coxsackie hybrid has been generated that contains the 220-625 nucleotides from coxsackie virus, replacing the 220-627 nucleotides of poliovirus (Semler et al., 1986) . This hybrid is a ts mutant for genome replication! One of the most salient features of the Y-end of picornavirus mRNA is that it does not contain the cap structure typical of eukaryotic mRNA . Instead, the viral RNA terminates in pUpU. Within the virion particle, or during the replication of the viral genome, this end is covalently bound, in the case of poliovirus, to a virus-encoded peptide of 22 amino acids Lee et al., 1977) . This peptide is the viral protein 3B and is also known as VPg (viral protein bound to the genome) (Nomoto et ai., 1977a, b) . Since picornavirus mRNA has no cap structure, the initiation of protein synthesis by these genomes is unusual . The initiation of translation in eukaryotic mRNAs commences by recognition of the cap structure by the initiator complex, a process involving cap-binding proteins (Kozak, 1989; . This recognition is followed by the attachment and scanning of the Y-end by the initiator complex, until an AUG is found in the correct context to start translation (Kozak, 1989; Cavener and Ray, 1991) . In picornavirus mRNA translation, the initiator complex binds, instead, to an internal region located between nucleotides 140-630 (Pelletier and Sonenberg, 1988) . In some picornaviruses, the initiator complex then scans the remaining Y-end until the initiator AUG is found, or it might even jump or translocate directly to this AUG . There is a pyrimide stretch UUUCCUUUU in this region, which is conserved in picornaviruses (Beck et al., 1983; from which nucleotides UUUCC (560-564) combined with the position of the seventh AUG located 150 nucleotides upstream of the initiator AUG are involved in the internal initiation of translation .

The 65 nucleotides located between the termination codons and the poly(A) tail play a role in the replication of viral RNA (Sarnow et al., 1990) . Indeed, a function of this region in the synthesis of negative-strand copies of RNA has been indicated . The presence of a pseudoknot in this region (Iizuka et aL, 1987; Jacobson et al., 1993) and the implication of these structures in the replication of viral RNAs has also been considered (Pleij, 1990) . The poly(A) tail plays a function in stabilizing mRNAs, and the infectivity of poliovirus RNA without this tail is reduced (Sarnow, 1989) . In addition, this poly(A) can participate in the replication of viral RNA (Richards and Ehrenfeld, 1990 ).

Once the ribosome starts translation at the initiator AUG, it travels through the 2209 codons of the poliovirus genome and generates a polyprotein Harber and Wimmer, 1993) . This polyprotein is not found as such in the infected cells, because it is cleaved while it is still on polysomes Lawson and Semler, 1990 ). The first cleavage is accomplished by the viral protease 2A, releasing the precursor (PI) of the capsid proteins (see the diagram depicted in Fig. 2 ) (Toyoda et al., 1986) . Once the other viral protease (protein 3C) has been synthesized, it cleaves between P2 and P3 (Section 4.1) (Hanecak et al., 1982) . Therefore, the viral proteases are active as precursors and are able to cleave themselves in cis. Indeed, once the mature proteases have been generated during infection, they are also active in trans Palmenberg, 1990; Lawson and Semler, 1990) . For more details on the proteolytic events and the specificity of these proteases, the reader is referred to several excellent reviews (Palmenberg, 1987 (Palmenberg, , 1990 Lawson and Semler, 1990; Hellen and Wimmer, 1992b) . Apart from the cleavages accomplished by the viral proteases 2A and 3C, a final cleavage event occurs during the assembly process on protein VP0 to generate VP4 plus VP2 (Palmenberg, 1987 (Palmenberg, , 1990 . This cleavage is catalyzed by VP0 itself, in conjunction with the viral RNA Palmenberg, 1987; Hellen and Wimmer, 1992b) .

In the case of poliovirus, 11 mature proteins plus a different array of precursors are produced, thus making the analysis of protein synthesis in picornavirus-infected cells very complex (Jacobson and Baltimore, 1968; Summers and Maizel, 1968) . Figure 3 shows the appearance of these polypeptides on two-dimensional polyacrylamide gels. The nomenclature of the picornavirus precursors and the mature proteins has been rationalized and is known as the L-4-3-4 nomenclature (Rueckert and Wimmer, 1984) . As indicated in Section 1.1, some picornaviruses encode for a protein, preceding the capsid proteins, that is known as L protein. Synthesis of L protein is followed by that of four capsid proteins (P1 region) and seven polypeptides involved in vegetative functions (P2 and P3 regions). These three regions in the genome, PI, P2 and P3, are distinguished according to the two proteolytic cleavages that take place on the polyprotein at the polysomal level. The mature proteins encoded in each of these regions are numbered 1, 2 and 3, followed by a letter A, B, C or D. Thus, the eleven mature proteins are known as 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C, 3D. The capsid proteins are also known by their original names: VP4 (or 1A), VP2 (or 1B), VP3 (or 1C) and VP1 (or 1D). Given the name of a viral protein, it is possible to identify its location on the genome. The precursor proteins are known by combinations of these names; for instance, precursor 3AB, 2BC or 2C3A. Although proteins 2C3A, or even 2B3A, are not found as such in infected cells, they can be generated by genetic engineering.

Picornaviruses possess small virion particles whose structure at atomic resolution has been solved (Hogle et al., 1985; Rossmann et al., 1985; Luo et al., 1987; Acharya et al., 1989; Kim et al., 1989; Rossmann and Johnson, 1989; Krishnaswamy and Rossmann, 1990; Yeates et al., 1991; Grant et al., 1992; Smyth et al., 1993) . The virions have a spherical shape and exhibit icosahedral symmetry (Rossmann and Johnson, 1989) . They are composed of 60 copies of each structural protein VP1, VP2, VP3 and VP4, plus a copy of the genome RNA that contains one copy of the genome-bound viral protein (VPg) . This genomic protein is covalently attached to the 5'-end of the viral RNA through an ester bond between the tyrosine present in VPg and the 5'-OH of the last nucleotide in the viral RNA (Lee et al., 1977) . The virion is composed of 60 identical asymmetric units, each formed by a copy of each of the structural proteins, although only three (VP1, VP2 and VP3) are on the virion surface, with VP4 being located internally (Hogle et al., 1985; Rossmann and Johnson, 1989) .

Determination of the structures of picornavirus proteins revealed that the virion proteins VP1, VP2 and VP3 have different amino acid sequences, but similar tertiary structures (Hogle et al., 1985; Rossmann et aL, 1985; Acharya et aL, 1989; Rossmann and Johnson, 1989; Grant et aL, 1992) . This tertiary structure is also shared by the single protein of the capsid of many plant RNA viruses, suggesting that a kind of basic structure or "brick" can exist within different virion particles (Rossmann and Johnson, 1989) . The protein fold that gives rise to this basic brick is depicted in Fig. 4 . It is composed of two four-stranded fl-sheets and is quite similar in different viruses (Hogle et al., 1985; Rossmann et al., 1985; Acharya et al., 1989; Rossmann and Johnson, 1989) . The minimum number of amino acids required to form a fl-barrel is ~ 150 (Rossmann and Johnson, 1989 ). There may be insertions of variable size between the strands at the cage of the traquetoid. VP1, VP2 and VP3 contain a common eight-stranded antiparallel //-barrel motif. Their amino termini intertwine to link them, thus forming a network in the interior of the capsid. Five VP3 amino termini form a five-stranded, helical/~-cylinder in the virion interior (Hogle et al., 1985;  (1985)). Rossmann et al., 1985; Acharya et al., 1989; Rossmann and Johnson, 1989) . VP4 is myristoylated at the amino terminus (Chow et al., 1987; Paul et al., 1987; Urzainqui and Carrasco, 1988) . This myristoylate moiety lies inside the virion coat near the fl-cylinder (Rossmann and Johnson, 1989) . On the surface of each protomer there is a depression of 25/~, depth, which is absent in aphtovirions (Hogle et al., 1985; Rossmann et al., 1985; Acharya et al., 1989; Rossmann and Johnson, 1989) . This depression or "canyon" has been implicated in the interaction of virion particles with receptors; neutralizing antibodies cannot enter into this depression (Rossmann and Johnson, 1989; Rossmann, 1989b; Olson et al., 1993a) (Fig. 5 ).

Viral infection commences by the recognition and attachment of virion particles to specific molecules located on the surface of cells, the so-called receptors (Dales, 1973; Dimmock, 1982; Lentz, 1990) . The chemical nature of the receptor molecules varies, depending on the species of virus that infects the cells, but many identified receptors are proteins and, more particularly, glycoproteins (Dales, 1973; Dimmock, 1982; Lentz, 1990) . In principle, a single virus species can use different receptor molecules on a single cell type or on different kinds of cells. In addition, different virus species can use the same receptor to land on the cell that is to become infected (Dales, 1973; Dimmock, 1982; Lentz, 1990) . The presence of receptors on the cell surface determines, in many instances, the viral tropism, i.e. the susceptibility of a given set of cells in the organism to infection by a particular virus (Dales, 1973; Dimmock, 1982; Lentz, 1990) . In addition, the interaction of viruses with cell surface receptors is one of the targets through which viral infection might be blocked (Lentz, 1990) .

In the case of picornaviruses, the receptor molecules for poliovirus and rhinovirus have been identified, the genes encoding these glycoproteins cloned and their protein products expressed and characterized (Greve et al., 1989; Mendelsohn et al., 1989; Staunton et al., 1989; Tomassini et al., 1989) . Both receptors belong to the superimmunoglobulin family of proteins (see reviews by Racaniello, 1990; Harber and Wimmer, 1993) . The poliovirus receptor is composed of three immunoglobulin-like domains located on the surface of the cell, a hydrophobic transmembrane domain that anchors the receptor to the membrane, and a cytoplasmic C-terminal region (Mendelsohn et al., 1989; Koike et al., 1990) (Fig. 6) .

The intercellular adhesion molecule-1 (ICAM-1) is the cellular receptor for rhinoviruses (Staunton et al., 1989 . This glycoprotein binds the lymphocyte function-associated antigen 1, an interaction involved in the immunological response (Staunton et al., 1989 , The receptor molecule ICAM-1 is composed of five immunoglobulin-like extracellular domains, a transmembrane domain of hydrophobic nature, and a short C-terminal cytoplasmic domain (Staunton et al., 1989 . Large amounts of truncated ICAM-1 lacking the hydrophobic and cytoplasmic domains have been obtained (Marlin et al., 1990) . These truncated forms are soluble and interact with rhinovirus particles (Marlin et al., 1990; Hoover-Litty and Greve, 1993; Olson et al., 1993a) . The structure of the truncated ICAM-I complexed with rhinovirus particles has been determined using cryoelectron microscopy, indicating that the receptor binds into the canyon (Olson et al., 1993a, b) .

The availability of cDNA clones coding for the poliovirus receptors made possible an analysis of the structure of the relevant gene and the transcripts derived from it in different cell lines (Koike et al., 1990 (Koike et al., , 1991a Morrison and Racaniello, 1992) . The poliovirus receptor gene contains eight exons, and its transcription produces a number of splice variants of mRNAs (Koike et al., 1990) . A duplication of the poliovirus gene has been found in monkeys, the two genes being encoded at different loci of the genome (Koike et al., 1992) . Mouse L cells are infected by poliovirus when transfected with the gene encoding the poliovirus receptor (Mendelsohn et al., 1989) . Transgenic mice have been generated carrying the poliovirus receptor gene (Ren et al., 1990; Koike et ai., 1991b; Ren and Racaniello, 1992) . These mice are susceptible to poliovirus and could constitute a good animal model to test the efficacy of poliovirus vaccines and the antiviral activity of poliovirus inhibitors in animals.

The binding of virion particles to their receptors induces conformational changes in some virion proteins that could trigger the interaction of these proteins with the membrane (Fricks and Hogle, 1990; Hoover-Litty and Greve, 1993) . Subsequently, the translocating activity of some of the virion proteins will be put in motion, thus inducing the passage of the virion through the lipid bilayer P6rez and Carrasco, 1993) . Some virion proteins are, therefore, endowed with translocation activity similar to that displayed by some domains of polypeptide toxins . However, this translocation capacity has not yet been assigned to any picornavirus protein.

In the model indicated above, receptor molecules are regarded as playing an active role during virus entry (Fricks and Hogle, 1990; Carrasco et al., 1993; Hoover-Litty and Greve, 1993) . The receptor binds the particle to the cell surface and this binding induces, in the virions, conformational changes that are required to uncoat the virion particle (Moore et al., 1990; Hoover-Litty and Greve, 1993 ) (see Section 2.3). This process is required to allow penetration of the membrane and entry to the cytoplasm (Fricks and Hogle, 1990; Hoover-Litty and Greve, 1993) . In agreement with this is the finding that binding of opsonized poliovirus to mouse L cells does not lead to infection (Mason et al., 1993) , indicating that the simple attachment of poliovirus to the cell surface does not suffice to initiate infection. Thus, the viral receptor in the cell plays a role beyond attachment (Mason et al., 1993) . Although lowering the pH in endosomes can contribute to the conformational changes that occur in virions, this process is not necessary in the ease of poliovirus (P6rez and Carrasco, 1993 ) (see Section 2.3). Even in some enveloped viruses that have recently been investigated, binding of enveloped virions to receptors also induces conformational changes in the viral glycoproteins that are responsible for membrane fusion (Moore et al., 1990; Meyer et al., 1992) . In principle, low pH would not be required to induce the conformational changes that lead to membrane fusion in the case of enveloped viruses, but would provide a proton-motive force to help the virion particle to trespass the membrane .

The mechanism used by animal viruses to enter cells and to deliver their genomes in the cytoplasm is a subject of intensive research (Hoekstra and Kok, 1989; Marsh and Helenius, 1989) . Animal viruses containing lipid membranes accomplish this task by fusion of the external envelope with cellular membranes, involving either the plasma membrane, as occurs with Sendai virus, herpesvirus or vaccinia virus, or the endosomal membrane, as is the case of semliki forest virus (SFV), vesicular stomatitis virus (VSV) or influenza virus (Hoekstra and Kok, 1989; Marsh and Helenius, 1989) . In the latter case, fusion is promoted by conformational modifications of the viral glycoproteins after receptor binding and acidification of endosomes (Wiley and Skehel, 1987; White, 1990) . Prevention of this acidification by lysosomotropic agents leads to inhibition of infection by SFV, VSV or influenza virus (Hoekstra and Kok, 1989) . Models that explain the entry of animal virus particles devoid of a lipid envelope, such as picornaviruses, are more scarce (Hoekstra and Kok, 1989; Marsh and Helenius, 1989) . In principle, these viruses could pass directly through the plasma membrane after receptor binding. Some evidence supports this mode of entry for certain viruses, such as adenoviruses (Brown and Burlingham, 1973) , poliovirus (Dunnebacke et al., 1969) , rotaviruses (Kaljot et al., 1988) and reovirus subviral particles (Borsa et al., 1979) . Alternatively, the viral particles can be internalized by receptor-mediated endocytosis and then cross the endosomal membrane (Everaert et al., 1989; Marsh and Helenus, 1989; Kronenberger et al., 1992) . In both cases, the viral genome must pass across a lipid membrane, aided by the virion proteins. Most evidence indicates that the majority of naked animal virus particles follow the receptor-mediated endocytosis pathway to infect cells (Marsh and Helenius, 1989) . Whether some of these viruses need a low pH step to trigger the crossing of the endosomal membrane, or enter cells by a pH-independent mechanism, remains open to debate (Brown and Burlingham, 1973; Madshus et al., 1984c; Svensson, 1985; Sturzenbecker et al., 1987; Gromeier and Wetz, 1990; Kronenberger et al., 1991; Varga et al., 1991) . Two major questions, therefore, remain to be answered with respect to the mechanism of picornavirus entry into cells: Is a given picornavirus species internalized through endosomes in a particular cell type?, or do endosomes need to be acidified for the virus to enter cells?

Attachment of poliovirus and its internalization in endosomes leads to conformational changes in the virion particle, particularly in VP1 (Fricks and Hogle, 1990) , that allow interaction with the membrane and aid the insertion of the myristoylated protein VP4 into the membrane (Lonberg Holm et al., 1975; Guttman and Baltimore, 1977; Fricks and Hogle, 1990 ). This insertion not only destabilizes the particle, but may also allow interaction with the membrane of some moieties of other viral proteins (Lonberg Holm et al., 1975; Guttman and Baltimore, 1977; Fricks and Hogle, 1990) (Fig. 7) . It can be speculated that the* insertion of the virion proteins into the membrane would open a pore through which the genome is delivered. Alternatively, energy may be required in the form of ATP, or as the proton-motive (membrane potential and/or pH gradient) generated in endosomes by the activity of the proton-ATPase pump, that could be used to drive the nucleocapsid through the lipid barrier of the membrane . A simple mechanistic model of viral particles opening pores in the membrane through which the genome passes is, however, inconsistent with the fact that the uncoating per se can occur in vitro in the presence of receptors (Hoover-Litty and Greve, 1993) , whereas the entry of the genome into the cytoplasm requires a metabolically active cell (Hoekstra and Kok, 1989; Marsh and Helenius, 1989) . The presence of ion channels in icosahedral virus particles (Kalko et al., 1992) may indicate that insertion of these capsids into membranes opens the ion channels that are required to dissipate the ionic gradients between endosomes and the cytoplasm. Fig. 7 . Two potential routes for picornavirus entry into cells and the co-entry of the toxin ct-sarcin. 1: the viral particle binds to its receptor and follows the endosomal route. 2: direct entry through the plasma membrane. The proton gradient generated by the vacuolar proton-ATPase is used by the virion proteins to translocate ~t-sarcin through the vacuolar membrane. 

One approach towards an understanding of the mechanisms by which animal viruses traverse the cellular membrane is to elucidate, in molecular terms, how the co-entry of protein toxins, or other macromolecules, and viral particles is achieved (Yamaizumi et al., 1979; Fernhndez-Puentes and Carrasco, 1980; FitzGerald et al., 1983; Carrasco et al., 1989) . The infection of susceptible cells by picornaviruses leads to profound changes in membrane permeability at two well-defined periods of the infection cycle: (1) early during infection when the virus enters the cells or (2) at the beginning of the late phase, before the onset of viral macromolecular synthesis takes place (for a review, see Carrasco et al., 1989) . This late modification of membrane permeability is also known as "late membrane leakiness" (Carrasco, 1978; Carrasco et al., 1989) . The molecular basis underlying both phenomena differs. Thus, only virion particles are necessary to accomplish the increase in membrane permeability early during infection (Fernfindez-Puentes and Carrasco, 1980; Carrasco, 1981) , whereas viral gene expression is necessary for the development of the late membrane leakiness (Carrasco, 1978; Contreras and Carrasco, 1979) . Selective compounds have been developed that block the replication of animal viruses during early and late modifications of the membrane (Carrasco and Vazquez, 1983) .

Fernfindez-Puentes and first observed that viral particles permeabilized cells to protein toxins that are otherwise unable to cross the membrane, because there are no receptors for them. The proteins are efficiently delivered into the cytoplasm shortly after addition of picornavirus to the medium. Indeed, almost 100% of the cells become permeabilized in a few minutes (Otero and Carrasco, 1987b; Lee et al., 1990) , suggesting that the viral particle contains a component that not only promotes the entry of the viral nucleocapsid into the cell, but also directs other macromolecules that are not physically bound to the particles towards the cytoplasm (Figs 7 and 8).

We now know that different protein toxins can cross the membrane in the presence of animal virus particles, and that other proteins, such as luciferase or horse-radish peroxidase (Otero and Carrasco, 1987b) , and polysaccharides (Gonzfilez et al., 1987) are also delivered into cells by these particles (Fig. 8) . No infectious virions are required for early membrane permeabilization to occur, because this phenomenon takes place with UV-inactivated (but not heat-inactivated) virions (Carrasco, 1981; Otero and Carrasco, 1987b; Carrasco et al., 1989) . More recently, the permeabilization of cells by adenovirus particles has been used to introduce DNA into cells. In a series of elegant experiments, Birnsteil and colleagues have shown that plasmids can bind to cells when they are complexed with transferrin-polylysine molecules (Zenke et al., 1990; Wagner et al., 1991) . The entry of these complexes is enhanced by more than 2000-fold when adenovirus particles are present in the culture medium. In this fashion, more than 90% of the cells express the transfected gene Wagner et al., 1992a, b; Zatloukal et al., 1992) .

We are starting to understand the exact mechanism by which the early membrane permeabilization of membranes, induced by viral particles, occurs. Certainly, receptor binding of poliovirus particles and virion uncoating are necessary for this process to take place (Almela et al., 1991) . It has been speculated that the delivery of protein toxins by animal viruses involves the disruption of endosomes (FitzGerald et al., 1983; Carrasco et al., 1989) , but other more selective mechanisms are possible. 0t-Sarcin has no effect on protein synthesis when added to uninfected HeLa cells, whereas the toxin efficiently blocks translation when added together with virion particles (Fern~mdez-Puentes and Carrasco, 1980; Otero and Carrasco, 1987b) . This co-internalization of the protein toxin by poliovirus is potently blocked by bafilomycin Al (BFLA1) (see Section 3.3.2), at concentrations that prevent infection with SFV or VSV (P6rez and Carrasco, 1993) .

These results indicate that an active vacuolar proton-ATPase is necessary for efficient delivery of 0t-sarcin into the cytoplasm mediated by poliovirus particles. We may conclude that most, if not all, of the toxin delivered into the cytoplasm comes from endosomal entry, because the inhibition of HeLa cells by ~t-sarcin was totally prevented by inhibiting the vacuolar proton-ATPase with BFLA 1 (P6rez and Carrasco, 1993) . A model of poliovirus entry illustrating the co-entry of ~t-sarcin and the site of action of different inhibitors is discussed in Section 3.4. Poliovirus enters from endosomes into the cytoplasm. The endosomal membrane remains intact because its rupture would lead to the eruption of the endosomal content, including ct-sarcin, into the cytoplasm. Although poliovirus permeabilizes cells to g-sarcin and this event occurs in endosomes, it is conceivable, although unlikely, that the virus has already released its genome into the cytoplasm after binding to the cell surface receptor, and that the virion proteins present in the membrane and internalized in endosomes are the ones that control permeabilization to g-sarcin (Fig. 7) .

Thus, the simple idea that enveloped animal viruses enter cells only by fusion between two membranes (Marsh and Helenius, 1989 ) is perhaps untenable, because such a model does not explain how the co-internalization of other macromolecules takes place (Yamaizumi et al., 1979; FitzGerald et al., 1983; Carrasco et al., 1989; Lee et al., 1990) . Perhaps the proton-motive force can be used to drive the viral nucleocapsid and other macromolecules through the lipid barrier of the membrane. Virion proteins may couple the energy liberated by ATP hydrolysis or the movement of protons and other ions into the cytoplasm in favour of its gradient to the translocation of the genome or protein toxins in the same direction (Carrasco et ai., 1993) .

3.1. Compounds that Bind to Virus Pro'titles As indicated in Section 2.1, on the surface of virion particles of rhinoviruses and poliovirus, there is a canyon crossing each protomer (Rossman, 1989b; Rossmann and Johnson, 1989; Harber and Wimmer, 1993) . Below this canyon there is a hydrophobic pocket within the VP1 fl-barrel, where a number of compounds that block virion uncoating bind (see reviews by Diana et al., 1985c; Rossman, 1989a; Rossmann and Johnson, 1989; McKinlay et al., 1992) (Fig. 5 ). This binding increases protein rigidity and stabilizes the particle in such a manner that uncoating cannot proceed (Rossmann, 1988; Chapman et al., 1991; Gruenberger et al., 1991; McKinlay et al., 1992) (Figs 5 and 9). Thus, these uncoating inhibitors act as "staples" on virion particles, stabilize them and block the disassembly of viruses.

The most refined studies on uncoating inhibitors have been carried out on a series of compounds developed by Sterling-Winthrop that are collectively referred to as WIN compounds ( Fig. 10 ) (Rossmann, 1989a; McKinlay et al., 1992) . Current knowledge on the interaction of these antivirai agents with rhinovirus particles has laid the groundwork for computer-assisted drug design .

One of the earliest steps during picornavirus uncoating is the formation of "A particles", which have a sedimentation coefficient of 125 S and have lost VP4 (Lonberg Holm et al., 1975; Guttman and Baltimore, 1977) . These particles are formed after infection of cells by picornaviruses or in vitro by exposure of virions to various treatments, e.g. altering the ionic conditions or the pH (Giranda et al., 1992) . The formation of these particles in vivo and in vitro is blocked by WIN compounds (Fox et al., 1986; Zeichhardt et al., 1987; Gruenberger et al., 1991) . Studies with human rhinovirus particles at atomic resolution (Rossmann et al., 1985) have aided the elucidation of the mode of interaction of a number of WIN compounds with these particles Badger et al., 1988 Badger et al., , 1989 Rossmann, 1988 Rossmann, , 1989a Kim et al., 1989 Kim et al., , 1993 Diana et al., 1990; McKinlay et al., 1992) . The binding site of these antiviral agents is in a hydrophobic pocket located beneath the canyon floor Rossmann, 1989a; Diana et al., 1990 Chapman et al., 1991; McKinlay et al., 1992; Kim et al., 1993; (see Figs 5 and 9). Binding of the drug to this pocket induces conformational changes in the floor of the canyon, causing it to be pushed upwards . This deformation can interfere with the binding of some rhinovirus serotypes with receptors , although other rhinovirus serotypes and poliovirus are not inhibited by WIN compounds with respect to their interaction with cell surface receptors (Fox et al., 1986; Zeichhardt et al., 1987; Almela et al., 1991) . Thus, some rhinovirus serotypes would be blocked at both the binding and uncoating steps (Shepard et al., 1993) , whereas inhibition of other picornaviruses by these drugs would be at the uncoating step only (Fox et al., 1986; Zeichhardt et al., 1987; Pevear et al., 1989; Almela et al., 1991) .

Rhinovirus 1A and polioviruses, in contrast with other rhinoviruses, bind a cofactor of lipidic nature in the pocket underneath the canyon floor (Filman et al., 1989; Yeates et al., 1991; Kim et al., 1993) . This cofactor is displaced after binding of WIN compounds, without alteration to the floor of the canyon (Kim et al., 1993) . This observation may explain why attachment of virion particles in the case of rhinovirus 1A and poliovirus is not blocked by WIN compounds (Kim et al., 1989) .

A number of clinical trials against common colds, using compounds that bind to rhinovirus particles and block uncoating, have been carried out (Tyrrell, 1984; Barrow et al., 1990; McKinlay et al., 1992; . Although results with dichloroflavan, Ro 09-0410 and 44,081 RP have been disappointing, recent trials with WIN 54954, R 77975 and R 61837 are more promising (see below). One of the major problems in the therapeutic treatment of common colds is to reach drug levels in the nasal epithelium that are sufficiently high to block viral growth (Reed, 1980; TyrreU, 1984) . WIN 51711 (disoxaril) was one of the first WIN compounds to be evaluated therapeutically (see below). The development of new derivatives with a broader spectrum of action and increased potency against rhinovirus prompted their clinical evaluation. WIN 54954 was not effective against rhinovirus when administered orally to a group of volunteers (Turner et al., 1993) . However, a prophylactic trial against the common cold caused by coxsackie A21 virus demonstrated the oral efficacy of this compound (McKinlay et al., 1992; Schiff et al., 1992 

Arildone (WIN 38020) and disoxaril (WIN 51711) ( Fig. 10 ) also belong to the group of WIN compounds. Arildone (4-6-(2-chloro-4-methoxyphenoxy)hexyl-3,5-heptanedione) is a selective inhibitor of poliovirus uncoating (McSharry et al., 1979; Caliguiri et aL, 1980; McSharry and Pancic, 1982; Eggers and Rosenwirth, 1988; Everaert et al., 1989) . Arildone and some related derivatives block the replication of several RNA and DNA viruses, including herpes simplex virus types 1 and 2 (Diana et al., 1977a, b; Kuhrt et al., 1979) , cytomegalovirus, SFV and VSV (Kim et al., 1980) . Administration of radioactive arildone to mice, dogs and monkeys by various routes indicates that it is extensively metabolized (Benziger and Edelson, 1982) . Arildone and the 3,5-dimethoxy isoxazole are active when administered orally in mice inoculated intracerebrally with poliovirus type 2 (Diana et al., 1985a) . The 3,5-dimethoxy isoxazole is more active against rhinovirus type 2 in cultured cells than is 4,6-dichloroflavan (Diana et al., 1985a) .

Disoxaril, 5-7-4-(4,5-dihydro-2-oxazolyl)phenoxy heptyl-3-methylisoxazole is a broad spectrum antipicornaviral agent (Diana et al., 1985a, b; McKinlay, 1985; Otto et al., 1985) . Oral administration of WIN 51711 was effective against echovirus and prevented poliovirus-induced paralysis and death in mice (McKinlay, 1985; McKinlay and Steinberg, 1986; McKinlay and Rossmann, 1989) . The W-2 strains of poliovirus type 2 can cause persistent infections in the CNS of immunosuppressed mice. Disoxazil treatment of these mice significantly decreased the incidence of disease and induced a rapid clearance of the virus from the CNS (Jubelt et al., 1989) . Unfortunately, side effects appear in patients when high oral doses of this compound are given .

Both of these compounds belong to a series of pyridazinamine analogues synthesized by Janssen Research Foundation (Fig. 10) . They block the replication of different rhin,'virus serotypes by stabilizing the viral particle and inhibiting uncoating (Andries et al., 1988; Rombaut et al., 1991) . Internalization of poliovirus particles into cells is not affected by R 78206, whereas uncoating and release of these particles into the cytosol are blocked (Rombaut et al., 1991; Ofori-Anyinam et al., 1993) . Assuming that these agents directly inactivate viral particles (Andries et al., 1988) and that mutants resistant to R 61837 are cross-resistant to other antiviral agents, including WIN 51711 , it is likely that these agents can bind into the same hydrophobic pocket, located beneath the canyon of human rhinoviruses, that is used by WIN compounds (Chapman et al., 1991) . Direct evidence for this interaction has been obtained by X-ray crystallographic analysis of human rhinovirus 14 complexed with R 61837 (Chapman et al., 1991) . This agent binds towards the pocket entrance, but fails to occupy the end of the pocket (Chapman et al., 1991) . It has been suggested that adding a tail to R 61837 to fit into the end of the pocket would improve the drug's activity against rhinovirus 14 (Chapman et al., 1991) . Clinical efficacy against the common cold was first demonstrated for R 77975 (pirodavir) (Hayden et al., 1992) . Administration of R 61837, applied as a nasal spray with hydroxy-fl-cyclodextrin as a vehicle, to human volunteers infected with rhinovirus 9 shows efficacy when given prophylactically, but not when used therapeutically (AI-Nakib et al., 1989; Barrow et al., 1990) .

SCH 38057 (1 [6-(2-chloro-4-methoxy)-hexyl]imidazole hydrochloride) (Fig. 10) is a water-soluble antiviral agent that binds irreversibly to enterovirus and rhinovirus particles (Zhang et al., 1993) . The structure of rhinovirus 14 complexed with SCH 38057 shows that this compound binds at the innermost end of the hydrophobic pocket of VP 1, leaving the entrance unoccupied. This interaction induces conformational changes involving a stretch of at least 36 amino acids of VP1 and VP3 (Zhang et al., 1993) . (Powers et al., 1982; Torney et al., 1982) . A number of phenoxybenzenes and phenoxypyridines were synthesized and evaluated for antiviral activity (Kenny et al., 1985) . Among these compounds, 2-(3,4-diChlorophenoxy)-5-nitrobenzonitrile was found to have a broadspectrum antiviral activity (Kenny et al., 1985) . The best antipicornavirus activity was found with 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridine carbonitrile (DEPC) (Kenny et al., 1985) . The uncoating of photosensitized coxsackie virus A21 was reduced by this compound (Kenny et aL, 1985) . Furthermore, DEPC has recently been shown to block the uncoating step in poliovirus (Gonzhlez et al., 1990a; Almela et al., 1991) .

A series of 2-phenyl-2H-pyrano[2,3-b]pyridines has been synthesized (Kenny et ai., 1986) , and some of them (MDL 20, 610, MDL 20, 646 and M DL 20, 957) (Fig. 11 ) showed potent antirhinovirus activity (Kenny et al., 1986) . The Y,4'-dichloro substitution, coupled with a 6-methylsulfonyl, 6-bromo or 6-chloro, is required for strong inhibition, and these compounds inhibit rhinovirus growth by 99% at concentrations of 4 ng/mL (Kenny et al., 1986) . Studies of their mode of action indicated that MDL 20,610 binds directly to rhinovirus particles and blocks uncoating (Kenny et al., 1988) .

A number of flavan derivatives (Fig. 11 ) were synthesized and assayed against rhinovirus type 1B (Bauer et al., 1981; Tisdale and Selway, 1983) . At a concentration of 7 nM, 4,ddichloroflavan (BW683C) blocked the replication of this rhinovirus in cultured cells, making this compound, at that time, the most active inhibitor of rhinovirus in culture systems (Tisdale and Selway, 1983 ). More recently, 3,5-dimethoxy isoxazole was shown to be more active than BW683C (Diana et al., 1985a) . Clinical trials with BW683C, given orally or intranasally, failed to protect from rhinovirus infections, despite the fact that the drug achieved good blood levels when given orally. However, it could not be found in nasal secretions (Phillpotts et al., 1983a; Al-Nakib et al., 1987) . Radioactive BW683C binds to rhinovirus particles and acts on an early step during infection (Bauer et al., 1981; Tisdale and Selway, 1983) . Several 4,6-dicyanoflavans based on BW683C have now been synthesized and give good inhibition not only of rhinoviruses, but also of other picornaviruses Desideri et al., 1990) . Finally, 1 -aniline-9-benzyl-2-chloropurines, containing a lipophilic para substituent, are good inhibitors of rhinovirus 1B (BW4294) (Kelley et al., 1990) .

Ro 09-0410 (4'-ethoxy-2'-hydroxy-4,6'-dimethoxychalcone) ( Fig. 11 ) is an active compound against rhinoviruses and binds to the viral capsid (Ishitsuka et al., 1982a, b; Ninomiya et al., 1984 , 1985, 1990) . Radioactive chalcone (Ro 09-0410) binds specifically to rhinovirus particles and this binding is inhibited by BW683C or RMI-15,731 (Ninomiya et al., 1985; Ishitsuka et al., 1986) . Of 53 rhinovirus serotypes tested, 46 were inhibited by Ro 09-0410 (Ishitsuka et al., 1982a) . Mutant rhinoviruses resistant to chalcone were cross-resistant to BW683C or RMI-15,731 (Ishitsuka et al., 1986) . These results indicate that the three compounds had a similar mode of action based on their binding to virion particles. Several analogues of Ro 09-0410 have been synthesized, some of them being 10 times more active than the parent compound (Ninomiya et al., 1990) . Administration of 1-2 ng/mL Ro 09-0696 is sufficient to significantly reduce rhinovirus replication in cultured cells (Ninomiya et al., 1990) . These analogues bind to the same site on the virus particles as Ro 09-0410 and have a similar mode of action (Ninomiya et al., 1990) .

3.1.9. 44081 RP 44081 RP, 2-(1,5,10,10a-tetrahydro-3H-thiaxolo 3,4-fl-isoquinolin-3-ylidine) amino-4-thiazole acetic acid (Fig. 12) , shows selective antirhinovirus activity (Zerial et al., 1985) and is active at concentrations lower than those that cause cytotoxic effects (125 #g/mL). The compound was not virucidal and acted on an early step in infection (Zerial et al., 1985) by blocking the uncoating of virion particles (Alarc6n et al., 1986) .

2-Thio-4-oxothiazolidine (rhodanine) (Fig. 12 ) selectively inhibits the uncoating of echovirus type 12 (Eggers, 1977) . The adsorption of virions to cells, and their subsequent entry into the cytoplasm, are not affected by this compound. Other picornaviruses and RNA-or DNA-containing viruses are not susceptible to inhibition by rhodanine. The compound has no deleterious effects on cell morphology or cellular macromolecular synthesis (Eggers, 1977; Eggers et al., 1979; Rosenwirth and Eggers, 1979) and protects the virion from both alkaline degradation (Eggers, 1977) and heat-inactivation (Rosenwirth and Eggers, 1979) . Rhodanine apparently blocks uncoating, since the compound prevents the shut-off of host protein synthesis induced by echovirus (Rosenwirth and Eggers, 1977) . It is well established that inhibition of host translation by picornaviruses requires translation of the input genomic RNA , a step that does not take place in the presence of uncoating inhibitors.

3.1.11. 731 RMI 15,731 (1-[5-tetradecyloxy-2-furanyl]-ethanone) ( Fig. 12 ) directly inactivates rhinovirus particles (Ash et al., 1979) . The mode of action of this agent has still not been investigated in detail. It is possible that the compound blocks uncoating (Ninomiya et al., 1985) , but it may have other effects because it was active if cells were pretreated and then washed, or if the agent was added 6 hr after infection (Ash et al., 1979) . The chemical structure of RMI 15,731 is similar to that of 5-(tetradecyloxy)-l-furoic acid (TOFA), an inhibitor of poliovirus genome replication (see Section 6.11).

There are several agents that interact with picornavirus particles and stabilize them against heat-inactivation and/or decapsidation. This is the case for L-cystine, but not for other amino acids (Pohjanpelto, 1958) , glutathione (Fenwick and Cooper, 1962) , 2-thiouracil (Steele and Black, 1967) and the methylthiopyrimidine derivative S-7 ( Fig. 12 ) (Yamazi et al., 1970; La Colla et al., 1972; Lonberg Holm et al., 1975) . Treatment of poliovirus with 1 mM 2-thiouracil resulted in direct inactivation of infectivity. Perhaps oxidized forms of this compound react with sulfhydryl groups of capsid proteins, inactivating them (Steele and Black, 1967) . S-7 inhibits the thermal inactivation of poliovirus particles and their decapsidation in HeLa cells (Lonberg Holm et al., 1975) .

Inhibition of virus attachment to its receptor results in a block of viral growth (Lentz, 1990) . This inhibition can be achieved by several means. One of them is to use antibodies against viral particles that block their interaction with receptors. Another approach is to antagonize the receptor by antibodies directed against the receptor itself (Minor et al., 1984; Colonno et al., 1986; Minor, 1990 Minor, , 1992 . Application of this latter approach in human volunteers infected with rhinovirus delayed the onset of symptoms of the disease, but not the infection (Hayden et aL, 1988) . Finally, anti-idiotypic antibodies directed against virions or viral receptor antibodies also inhibit viral infection (Lentz, 1990) .

Another approach to blocking poliovirus or rhinovirus infections uses soluble receptors, i.e. moieties of receptor molecules devoid of the carboxy terminus responsible for anchoring the molecule to the membrane (Kaplan et al., 1990a, b; Marlin et al., 1990; Hoover-Litty and Greve, 1993) . Incubation of virus particles with their receptors or truncated (soluble) receptors leads to the formation of complexes and the selective inhibition of infection (Kaplan et al., 1990a, b; Marlin et al., 1990; Hoover-Litty and Greve, 1993) . Chimeric molecules of ICAM-1 and immunoglobulins are more effective in inhibiting rhinovirus binding to cells (Martin et al., 1993) . The utility of this approach for attacking viral infections in the organism is limited since, for example, mutant viruses that are resistant to neutralization by the receptor readily arise (Kaplan et al., 1990b) .

Amines are thought to accumulate in endosomes and other organelles of the vesicular system, where they become protonated. Accordingly, they permeate less readily and, thus, cause a rise in the endosomal pH (Fletcher et al., 1965; Seglen, 1983) . In contrast, monensin and nigericin ( Fig. 13 ) are ionophores that exchange Na ÷ or K÷/H + in favour of an H + gradient, thus dissipating the pH gradient created by the proton-ATPase pump (Seglen, 1983) . As indicated above, there are, in principle, two possible mechanisms of poliovirus entry, i.e. direct entry through the plasma membrane (Dunnebacke et al., 1969) or internalization in endosomes (Zeichhardt et al., 1985) . Poliovirus might use both of these mechanisms in different cell types or even in the same kind of cell. Once the viral particles are in endosomes, they must exit from this organeile in order to initiate translation of the genomic RNA in the cytoplasm. It is still a matter of debate as to whether poliovirus requires acidification of endosomes to allow the passage of the genome through the endosomal membrane (Madshus et al., 1984b, c; Zeichhardt et al., 1985) , or if poliovirus accomplishes this task in a pH-independent fashion (Gromeier and Wetz, 1990; P~rez and Carrasco, 1993) . Indirect experiments involving complexing of poliovirus particles with neutral red prior to incubation for several minutes with weak amines or monensin, followed by very long incubation (18-40hr), have led to the suggestion that poliovirus requires a low-pH step for infectivity (Madshus et al., 1984b, c) . Virus entry was evaluated by measuring the cytopathic effect caused by poliovirus complexed with neutral red after several rounds of viral replication (Madshus et al., 1984b, c) . High concentrations of amines have also been used, e.g. 0.3 mM chloroquine, or 70 mM NH4CI (Zeichhardt et al., 1985) . Poliovirus polymerase production was strongly inhibited, even when the compounds were added 1 or 2 hr after virus entry (Zeichhardt et al., 1985) . Certainly, the lysosomotropic agents employed have a number of side effects that influence a variety of enzymes and functions apart from raising the endosomal pH (Seglen, 1983; Hiebsch et al., 1991) . Hence, it is not surprising that during the long incubation times, where several rounds of viral replication take place, there was inhibition of the virus (Madshus et al., 1984b, c) . Shorter incubation times testing the inhibition of poliovirus by chloroquine or monensin during the uncoating period lead to the idea that poliovirus entry is pH-independent (Gromeier and Wetz, 1990; P6rez and Carrasco, 1993) . The presence of lysosomotropic agents, such as chloroquine, amantadine, dansylcadaverine or monensin, during poliovirus entry did not inhibit infection (P~rez and Carrasco, 1993) .

Studies on the requirement of a low-pH step for the entry of other members of the Picornaviridae family indicate that replication of FMDV is inhibited by lysosomotropic compounds (Carrillo et al., 1984 (Carrillo et al., , 1985 Baxt, 1987) . A direct action of these agents in the initial steps of virus infection is still uncertain; however, chloroquine was still active in the inhibition of FMDV replication, even when added 2.5 hr after infection (Baxt, 1987) , whereas monensin (Neubauer et al., 1987) , as well as BFLA1 (P~rez and Carrasco, 1993) , blocked the internalization of labelled rhinovirus particles into HeLa cells. It has been claimed that monensin might even increase infection of cells by EMC virus when low-pH medium is used during virus penetration (Madshus et al., 1984a) . Entry of EMC virus into HeLa cells is not inhibited by increasing the pH in endosomes (P~rez and Carrasco, 1993) . Fig. 13 . Chemical structures of different compounds: bafilomycin AI, monensin, chloroquine and nigericin.

HO Nigericin The producer organism, the molecular mass (MW), number of amino acids (AA) and the mode of action are indicated.

Three major classes of proton-ATPases exist in mammalian cells: (1) the mitochondrial F-class ATPases, (2) the plasma membrane proton ATPases of the P-class and (3) the V-class of vacuolar proton-ATPases (Schneider, 1987; Nelson and Taiz, 1989) . Each class is distinguished by several parameters, including their sensitivity to inhibitors (Nelson and Taiz, 1989) . Endosomes, along with other organelles of the vesicular system, become acidified by the action of the vacuolar proton-ATPases (Schneider, 1987; Nelson and Taiz, 1989) . These enzymes pump protons into the organelles at the expense of ATP hydrolysis (Schneider, 1987; Nelson and Taiz, 1989) . The two major classes of inhibitors, hitherto used to assess the requirement for a low-pH step during virus infection, are either weak amines (NH4CI, chloroquine, amantadine, etc.) that raise the endosomal pH by their accumulation of ionophores (monensin, nigericin) that dissipate the proton gradient (Seglen, 1983 ) (see Section 3.3. I). In some studies, the inhibitor dicyclohexylcarbodiimide (DCCD), which blocks all types of proton-ATPases, has been assayed (Seglen, 1983; Hiebsch et al., 1991) . BFLA1 (Fig. 13) , a recently discovered antibiotic, is a powerful and selective inhibitor of the vacuolar proton-ATPases (Bowman et al., 1988; Nelson and Taiz, 1989; Zeuzem et al., 1992; Dr6se et al., 1993) . BFLA1, in contrast to DCCD, which nonspecificaUy blocks all types of proton-ATPases (Nelson and Taiz, 1989) , is a unique compound for assaying the roles that acidification of endosomes and the action of the vacuolar proton-ATPase play in the entry of animal viruses. Viruses such as SFV or VSV, which enter cells through endosomes and need their acidification, are potently inhibited by BFLA1, whereas poliovirus infection is not affected by the antibiotic (Prrez and . Therefore, the entry of poliovirus is not restricted by the three different techniques used to increase the endosomal pH, i.e. (1) the blockade of the vacuolar proton-ATPase with BFLA1, (2) the raising of the endosomal pH by weak amines and (3) the dissipation of the pH gradient with monensin. The effect of BFLA 1 on other members of the Picornaviridae family indicates that entry of EMC virus into HeLa cells was not affected by this macrolide antibiotic, whereas rhinovirus was sensitive to its action (Prrez and . The co-entry of toxins, such as a-sarcin, together with poliovirus particles is potently inhibited by BFLA1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization induced by poliovirus (see Section 2.4).

A number of protein toxins are effective inhibitors of protein synthesis (V~tzquez, 1979; Jimenez and V~tzquez, 1985) (Table 1 ). The mode of action of these toxins in intact cells involves two steps, namely attachment of the toxin to cell membrane receptors and entry of the toxin, or its effector moiety, into the cytoplasm where enzymatic inactivation of the components of the protein synthesizing machinery occurs. Some toxins, such as diphtheria or Pseudomonas exotoxin A, inactivate the elongation factor EF-2 by ADP-ribosylation (Aktories, 1992) , whereas ricin, abrin, ct-sarcin, mitogillin, etc., inactivate ribosomes by acting on the 28S rRNA Endo and T'surugi, 1987; Hartley et al., 1991; Better et al., 1992; Irvin and Uckun, 1992) . Some plant toxins, such as mitogillin, restrictocin or ct-sarcin, do not bind to receptors, but are potent inhibitors of translation in cell-free systems or in permeabilized cells (Jimenez and V~tzquez, 1985; Carrasco and V~izquez, 1983) .

Poliovirus is able to co-internalize, and, thus, deliver, protein toxins efficiently into cells (Carrasco, 1981; Carrasco et al., 1989; Lee et al., 1990; Almela et al., 1991) . The antiviral potency of these toxins is high because after the toxin has entered along with the infecting virus, the translation of viral proteins does not occur and the formation of the progeny virus is strongly suppressed (Ussery et al., 1977; Yamaizumi et al., 1979; Fernandez-Puentes and Carrasco, 1980; Carrasco, 1981; Carrasco and Esteban, 1982; Irvin and Uckun, 1992) . In addition to protein toxins, low molecular weight antibiotics also enter cells early during infection (Carrasco, 1981; Carrasco and V~izquez, 1983) . In general, hydrophilic antibiotics are selective inhibitors of viral translation (Carrasco and V~izquez, 1983) , thus blocking viral replication at concentrations that do not affect uninfected cells (Lacal and Carrasco, 1983b ) (see also Section 4.3.1). The mode of action of different inhibitors on the entry of poliovirus and the co-entry of toxins is illustrated in Fig. 14.

Each picornavirus possesses a single species of mRNA that is identical to its genome . This long RNA (7.2-8.5 Kb) has a single initiator AUG ) except for FMDV, where initiation can take place on two different AUGs that are in phase and direct the synthesis of two different L proteins (Belsham, 1992 of picornavirus RNA is accomplished by the host protein synthesizing machinery using, in general, the same mechanism as that used to translate any cellular mRNA, particularly with respect to the elongation and termination phases. However, the initiation of translation of picornavirus RNA shows some unusual features that will now be considered. All known cellular mRNAs possess a cap structure at their 5'-end that is recognized by the translation initiation complex (Rhoads, 1988 (Rhoads, , 1993 . Once the ribosomal 40S subunit plus a number of initiation factors are bound to this capped 5'-end, they scan the mRNA leader sequence until an AUG is found in the correct context to start translation (Sonenberg, 1990; Kozak, 1992) . This mechanism is not used for the initiation of picornavirus translation, because the initiation complex binds to a unique internal sequence within the long leader sequence (Sonenberg, 1990) . In poliovirus, this region is located approximately between nucleotides 134 to 585-612 of the leader sequence (Sonenberg, 1990; Kozak, 1992) . The secondary structure of the 5' leader region of different picornaviruses reveals two different patterns, one for entero-and rhinoviruses and another for cardio-and aphtoviruses (Jang et al., 1988; Pelletier and Sonenberg, 1988; Belsham, 1992; Harber and Wimmer, 1993) (Fig. 15) . It is even possible that the requirements and mechanism of initiation on the RNAs of these two classes of viruses differ . Thus, the initiation of translation on the poliovirus mRNA can start by the attachment of the initiation complex to this internal region of the leader sequence, which has been christened as the ribosome landing pad (RLP) or internal ribosome entry site (IRES). This event could be followed by a scanning phase for selection at the initiator AUG (Sonenberg, 1990; Kozak, 1992) . In contrast, initiation on EMC virus RNA commences by the direct binding of the initiator complex to the IRES (or RLP) and to the initiator AUG. A scanning phase does not appear to be involved .

Since picornavirus RNA has a high binding affinity for ribosomes, it exhibits different requirements for translation with respect to initiation factors. Picornavirus RNA was the first mammalian mRNA to be translated in cell-free systems (Kerr and Martin, 1971) , and these studies showed that its translation required different concentrations of some initiation factors when compared with translation of cellular mRNAs (Wigle and Smith, 1973) . More recent studies have analyzed the effects of exogenously added individual initiation factors to rabbit retieulocyte lysates programmed with capped or uncapped mRNAs. Internal initiation on synthetic bicistronic mRNAs that contain the poliovirus 5'-end in the second cistron is increased by addition of elF-4B and elF-4F (Anthony and Merrick, 1991) , indicating that elF-4F participates in the translation of naturally uncapped mRNAs (Anthony and Merrick, 1991; Thomas et al., 1991) .

Apart from the initiation factor requirement for efficient translation, a number of cellular proteins with binding affinity for picornavirus RNA have been identified, but the roles that these proteins could play during internal initiation of translation, or as "initiation correction factors", deserve further investigation . Viral proteins can also bind to specific regions on the 5'-end of mRNA and, perhaps, might affect translation. This is the case for poliovirus protein 3CD that binds to the 5'-end (Andino et al., 1990a) , or for protein 2A, which enhances translation of poliovirus RNA by an as yet unidentified mechanism (Hambidge and Sarnow, 1992) .

One of the most interesting aspects of the translation of picornavirus RNAs is that, in comparison with cellular mRNAs, they require a higher concentration of monovalent cations for optimal translation (Carrasco and Smith, 1976) . In fact, under the ionic conditions required to initiate translation efficiently on picornavirus RNA, the initiation on cellular mRNAs is restricted (Saborio et al., 1974; Carrasco and Smith, 1976; Carrasco et al., 1979) . The discrimination between viral and cellular mRNAs shown by monovalent cations in cell-free systems is such that at higher cation concentrations, the resulting effects perfectly mimic the shut-off of host translation observed in intact cells after infection (Carrasco and Smith, 1980) . The physiological significance of these requirements is related to the fact that viral protein synthesis takes place mainly in a cytoplasm where monovalent ion concentrations have drastically changed (see review by Carrasco et al., 1989) as a consequence of membrane leakiness produced during picornavirus infection. This observation explains the shut-off of host translation during infection that is observed in some members of the Picornaviridae family (Carrasco, 1977 (Carrasco, , 1987 Carrasco and Castrillo, 1987) .

Picornaviruses encode for the proteases responsible for the processing of the polyprotein to produce mature viral proteins (Krausslich and Hellen et al., 1989; Lawson and Semler, 1990; Palmenberg, 1990) . The picornavirus proteases have been classified as cysteine peptidases (Rawlings and Barrett, 1993) . The two poliovirus proteases, proteins 2A and 3C, are active in the precursor form and are able to cleave substrates in c/s. Indeed, after their proteolytic modification, they can recognize their substrates in trans even in the nascent polypeptide chain. Viral proteases are highly specific and cannot be interchanged between different picornavirus species Hellen et al., 1989; Lawson and Semler, 1990; Palmenberg, 1990) . Apart from cleaving the viral polyprotein and the polypeptide precursors, they can also cleave a limited number of cellular proteins (Grigera and Tisminetzky, 1984; Urzhinqui and Carrasco, 1989; Falk et al., 1990; Clark et al., 1991; Joachims and Etchison, 1992) . The best illustrated example is the cleavage of polypeptide p220 that forms part of the translation initiation factor elF-4F induced by poliovirus protease 2A pr° (Etchison et al., 1982; Lloyd et al., 1988) . Cleavage of p220 is not directly accomplished by 2A pr°, but by another cellular protease that is activated by 2A pr° (Wyckoff et al., 1990 (Wyckoff et al., , 1992 . It was suggested that cleavage of p220 was a crucial step in the inhibition of host translation by poliovirus infection (Etchison et al., 1982) . However, recent evidence indicates that this may not be so. Thus, there is no correlation in time between p220 cleavage and the shut-off of host translation (Etchison et al., 1982; Bonneau and Sonenberg, 1987; Ptrez and Carrasco, 1992) ; the presence of some inhibitors of poliovirus RNA synthesis allows complete cleavage of p220, while for hours there is still a substantial amount of host protein synthesis (Bonneau and Sonenberg, 1987; Ptrez and Carrasco, 1992) ; translation of several capped viral mRNAs can take place in poliovirus-infected cells (Alonso and Carrasco, 1982b; M ufioz et al., 1985a; Castrillo and Carrasco, 1987b; Schrader and Westaway, 1990 ); elF-4E, an initiation factor that, together with p220 forms elF-4F, is localized in the nucleus and not in the cytoplasm (Lejbkowicz et al., 1992) ; monocytic cell lines persistently infected with poliovirus synthesize proteins even though p220 has been extensively, but not completely, degraded (Lloyd and Bovee, 1993) ; a poliovirus mutant in 2A that is unable to cleave p220 still shuts down host translation (Bernstein et al., 1985) ; elF-4F stimulates the translation of capped or naturally uncapped viral mRNAs (Anthony and Merrick, 1991; Thomas et al., 1991) ; transient expression of poliovirus protein 2A, the protease that induces p220 cleavage, has a major impact on transcription of a reporter gene, compared with its translation ; finally, disruption of the yeast p220 gene homologue does not block cellular growth (Lanker et al., 1992) .

Precise action of picornavirus proteases is vital for generating the mature viral proteins that are, in turn, required for a number of viral processes, including replication of the picornavirus genome (Krausslich and Hellen et al., 1989; Lawson and Semler, 1990; Palmenberg, 1990 ). Thus, it seems feasible that selective interference with protease function would lead to the specific inhibition of viral growth (Korant et al., 1986; Hellen and Wimmer, 1992a) . Indeed, a number of agents have been described as inhibitors of protease function (Korant et al., 1986; Korant, 1987 Korant, , 1990 Korant, , 1991 and can be broadly classified in two categories: (1) compounds that modify the conformation of the viral polyprotein and render it refractory to protease action and (2) inhibitors of the proteases themselves.

With respect to the first category, a number of amino acid analogues (Jacobson et al., 1970; Jacobson and Baltimore, 1968) , zinc ions (Korant et al., 1974) or high temperature lead to conformational changes of the poliovirus polyprotein, which is then no longer recognized by viral proteases (Korant, 1990 (Korant, , 1991 . However, this approach is not selective for inhibition of viral proteases since cellular proteins are also affected.

A number of amino acid analogues have been used to inhibit the proteolytic degradation of the picornavirus polyprotein. In fact, these agents were employed to identify picornavirus precursors and to block the formation of mature proteins. Compounds such as DL-ethionine (Brown and Ackermann, 1951) , TPCK (tolylsulfonylphenylalanyl chloromethyl ketone), TLCK (tolylsulfonyl lysyl chloromethyl ketone) (Korant, 1972) , ZPCK (carbobenzyloxyphenylalanyl chloromethyl ketone) (Summers et al., 1972) , ZLCK (carbobenzoxyleucyl chloromethyl ketone) (Korant et al., 1979) , canavanine or fluorophenylalanine (Brown et al., 1961; Levintow et al., 1962; Scharff and Levintow, 1963) (Fig. 16) inhibit the proteolytic processing of the picornavirus polyprotein.

The activities of cysteine proteases are controlled by a set of protein inhibitors that constitute the cystatin superfamily of proteins. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. Cystatins are small proteins (110-120 amino acids) that bind and selectively inhibit cysteine proteases. Human cystatin C, obtained from human serum, and human stefin B, obtained from spleen, also bind and inhibit poliovirus protease 3C and block the replication of poliovirus in cultured cells (Korant et al., 1985 (Korant et al., , 1986 . Not only cystatin C, but also peptides that mimic the proteinase-binding center of cystatin C, such as Z-LVG-CHNHz (N-benzyloxycarbonylleucyl-valyl-glycine diazomethylketone), show antiviral activity against poliovirus and herpes simplex virus (see Section 4.2.2) (Bjorck et al., 1990) .

The use of peptides that mimic the viral cleavage site provides a potential for the development of new protease inhibitors, because they inhibit protease function (Green and Shaw, 1981) . Therefore, analogues of the recognition site of viral proteases have a modified C-terminal or the N-terminal blocks viral replication (Bjorck et al., 1990; Korant, 1990) . Tri-, tetra-or pentapeptides having, in the C-terminal, chloromethyl ketone or methyl ketone moieties, were active against poliovirus and rhinovirus (Kettner and Korant, 1987) .

The thiol protease inhibitor E-64 (Fig. 16) is a natural product isolated from Aspergillus japonicus. It has also been chemically synthesized. E-64 and leupeptin are inhibitors of the cysteine protease calpain (Mehdi, 1991) . E-64 derivatives lacking charged groups inhibit intracellular proteases (Mehdi, 1991) . E-64 specifically blocks the L-protease of FMDV, leading to a reduction in virus yields (Kleina and Grubman, 1992) . Addition of a permeable analogue to cultured cells infected with FMDV decreases viral RNA synthesis, cleavage of p220 and retardation of the virus-induced shut-off of host translation (Kleina and Grubman, 1992) . It is not yet known whether this last effect is due to inhibition of the L-protease itself or to the general retardation of viral replication. E-64 and its permeable derivatives are nontoxic in animals, and doses of 400 mg/kg of body weight are well tolerated by various administration routes (Komatsu et al., 1986; Kleina and Grubman, 1992) . 

A series of spiro indolinone fl-lactams have been synthesized and their activity assayed against poliovirus and rhinovirus 3C proteases (Skiles and McNeil, 1990) . The sulfonamide derivative shown in Fig. 16 was active not only against these picornavirus proteases, but against other cellular proteases such as human leukocyte elastase and cathepsin G (Skiles and McNeil, 1990) . Thysanone (Fig. 16) is a novel naphthoquinone isolated from the fungus Thysanophora penicilloides, which is active against rhinovirus 3C protease (Singh et al., 1991) . The selectivity of these compounds in cell culture has yet to be investigated.

Although viral proteases have repeatedly been suggested as potentially effective targets for the inhibition of viral growth (Korant, 1987; Hellen and Wimmer, 1992a) , no clinical results have been reported to support this premise.

The most drastic modifications of membrane permeability occur in the late phase of the replication cycle of many cytolytic viruses, and a detailed review on this subject has already been published (Fig. 17) . The major salient feature is that a viral gene product is required for the development of late modifications in membrane permeability. This modification starts in HeLa cells at about the third hour after infection with poliovirus (Nair et al., 1979; Lacal and Carrasco, 1982; Mufioz and Carrasco, 1983; Nair, 1984; L6pez-Rivas et al., 1987; Garry, 1989) , or at the second hour p.i. in cells infected with SFV (Garry et al., 1979; Ulug et al., 1987) . Thus, the majority of viral macromolecules are synthesized in cells with an altered plasma membrane in a cytoplasm where the concentrations of ions, and perhaps pH and divalent cations, are gradually changing (Carrasco and Smith, 1976; Mufioz et al., 1985a, b; L6pez-Rivas et al., 1987; Holsey et al., 1990) . The mechanism of membrane permeabilization by poliovirus is fairly well understood. There is an overall increase in permeability to a variety of molecules , and the modification of the membrane is such that the gradients of monovalent cations are destroyed. In addition, other metabolites and compounds that are excluded by the membrane leak out from cells and can readily pass to the cytoplasm (Egberts et aL, 1977; Benedetto et al., 1980; Nair, 1981 ).

An updated view of the modifications of membrane permeability by animal viruses is shown in Fig. 17 . The putative viral protein responsible for membrane modification associates with membranes once it is synthesized and travels through the vesicular system to the plasma membrane. As we have already indicated, these viral proteins may possess an ionophore-like activity (Carrasco, 1977 (Carrasco, , 1987 Carrasco et al., 1993) , such that the pH and ionic content of vesicles of the vesicular system will be modified. Once these proteins reach the plasma membrane, they increase the permeability to ions, leading to the disruption of the membrane potential Carrasco, 1982, 1983a) . We have suggested that these changes of ions and pH in the cytoplasm influence an array of cellular functions, including the translation of host mRNAs (Carrasco, 1977 (Carrasco, , 1987 Carrasco et al., 1993) . With respect to the mechanism of late membrane permeabilization, we have found that phospholipase A2 activity is inhibited early following poliovirus infection, whereas the phospholipase C that hydrolyses phosphatidylinositol is stimulated from the third hour p.i. (Guinea et al., 1989) . Thus, in poliovirus-infected cells, the amount of inositol triphosphate (IP3) present in the cytoplasm constantly increases (Guinea et al., 1989) . This increase would lead to an augmentation of free calcium. However, we are not aware of any analysis of free calcium levels in cells infected by picornaviruses. Recently, we have analyzed in detail the activation of the phospholipase C that hydrolyses phosphatidylcholine, and found that both choline and phosphorylcholine drastically increase in the medium of poliovirus-infected cells from the third hour of infection .

It is clear that a correlation in time exists between the increase in membrane permeability and the activation of phospholipase C in poliovirus-infected cells (Guinea et al., 1989; Irurzun et al., 1993) . However, we do not know yet if there is a cause-effect relationship between them. A number of protein toxins, such as melittin, that modify membrane permeability also activate phospholipase activity (Durkin and Shier, 1981; Pellkofer et al., 1982) . It is tempting to speculate that poliovirus encodes a protein responsible for modifying the membrane (Lama and Carrasco, 1992a, b; Lama et al., 1992) . This protein could be poliovirus protein 3A, a membrane protein that contains an amphipathic helix. A similar motif is found in other viral membrane proteins that modify membrane permeability (Pinto et al., 1992; Carrasco et al., 1993) . Such viral proteins that are responsible for increasing membrane permeability have been collectively referred to as viroporins . The accumulation of a sufficient number of copies of such a viroporin would form pores in the membrane that may lead per se to the permeabilization of the membrane. A secondary effect could be the activation of phospholipase C, to generate diacylglycerol, which, in turn, also increases membrane permeability (Felix, 1982; Otero and Carrasco, 1988 ).

In principle, translation inhibitors that have their target on components of the protein synthesizing machinery would block viral and cellular translation nonspecifically. The action of compounds such as puromycin (Levintow et al., 1962) or pactamycin (Summers and Maizel, 1971; Taber et al., 1971) have been used to study the dependence of viral RNA synthesis on viral translation (Levintow et al., 1962) or to map the viral proteins on the picornavirus genome (Lawson and Semler, 1990) . However, there is no selectivity in this inhibition compared with the blockade of host protein synthesis in uninfected cells. A selective way to block protein synthesis in animal virus-infected cells was devised based on modification of membrane permeability during virus infection. This approach employs the use of hydrophilic translation inhibitors that do not easily cross the membrane barrier of normal uninfected cells, but readily enter into a number of animal virus-infected cells (Carrasco, 1978; Contreras and Carrasco, 1979; V~izquez, 1979; Carrasco et al., 1989) . These studies showed that the bulk of picornavirus protein synthesis occurs in cells where the membrane has been drastically modified . The presence of these translation inhibitors not only blocked selectively the synthesis of viral proteins, but also blocked the formation of new infectious virus, indicating that virus assembly takes place largely after the membrane has been modified (Benedetto et al., 1980) . Hence, inhibitors of translation that are hydrophilic and do not inhibit protein synthesis efficiently in intact cells are able to selectively block translation in a variety of animal virus-infected cells (Carrasco, 1978 used as a test to measure changes in membrane permeability not only in cells infected with animal viruses, but also in any cell type (eukaryotic or prokaryotic) where membrane permeability has been altered Carrasco, 1981a, b, 1982a; Otero and Carrasco, 1987a; Lama and Carrasco, 1992b) . The formation of infectious virus by these agents is strongly diminished at drug concentrations that do not affect translation in uninfected cells (Benedetto et al., 1980; Lacal et al., 1980; Lacai and Carrasco, 1983b) . Therefore, cell killing by low multiplicities of poliovirus is prevented by treatment of cell monolayers with hygromycin B (Lacal and Carrasco, 1983b) . This selectivity is not achieved by other inhibitors of translation that enter equally well in virus-infected or uninfected cells (Contreras and Carrasco, 1979; Lacal and Carrasco, 1983b) . This approach has not been exploited in animals because some of the nonpermeant antibiotics that block translation are not very potent, as occurs with guanylyl methylene diphosphate (Dawson et al., 1979) , or showed toxicity (unpublished results), perhaps due to different cellular susceptibilities in the animal after prolonged treatment. However, the approach of using inhibitors of any viral or cellular function that selectively enter into virus-infected cells early or late during infection, as a result of modification of membrane permeability, remains open. In other words, selectivity can be conferred on antiviral compounds by adding charged residues to make them less permeable in uninfected cells, whereas these charged molecules will readily enter virus-infected cells. In fact, we have observed that highly charged compounds, such as sulphated polysaccharides, suramin or the dye trypan blue, show good antiviral activity (Alarc6n et al., 1984c) , and the entry of sulphated polysaccharides into cells is enhanced by poliovirus particles (Gonzfilez and Carrasco, 1987 ) (see Section 2.4). These agents have been used by a number of laboratories in tests for antiviral effects against several animal viruses, including human immunodeficiency virus (HIV) (Mitsuya et al., 1984 (Mitsuya et al., , 1988 Balzarini et al., 1986; Gonzfilez et al., 1987; Zuckerman, 1987; Schols et al., 1990; Callahan et al., 1991; McClure et al., 1991) .

Once the viral genome is liberated in the cytoplasm after poliovirus entry, it is recognized by the ribosomes and the translation machinery as mRNA (Sonenberg, 1990) . Translation of the input viral RNA gives rise to a number of virus-encoded proteins that are necessary to replicate the genome Semler et al., 1988; Richards and Ehrenfeld, 1990) . Although many details of the steps involved in the replication of poliovirus RNA have been unravelled, this is perhaps one of the least understood processes in molecular terms. This is particularly so for the initiation of new RNA chains, the possible role played by some cellular proteins and the requirement of membranes in genome replication Semler et al., 1988; Richards and Ehrenfeld, 1990) . The replication of an RNA involving only RNA, and not DNA, intermediates, as occurs with retroviruses or hepadnaviruses, is perhaps one of the most primitive processes in the multiplication of biological molecules. An understanding of how picornaviruses accomplish their replication can shed light on the essential requirements of one of the most primitive forms of life and elucidate how other more complex RNA-containing viruses are able to replicate their genomes in infected cells. In addition, once the requirements for the replication of an RNA genome have been defined, they can be used for the amplification of any kind of RNA molecule, either in intact cells or in cell-free systems, as already achieved with phage RNA (Wu et al., 1992) .

One of the essential steps required for the amplification of a positive-stranded RNA molecule is the copying of this RNA to its complementary chain that, in turn, will be used as a template for synthesis of more positive-strand RNA molecules Semler et al., 1988; Richards and Ehrenfeld, 1990) (Fig. 20) . Thus, in picornavirus-infected cells, the only two classes of RNA molecules present are: the genomic positive-strand RNA or an exact copy of this RNA, the negative-strand RNA Semler et al., 1988; Richards and Ehrenfeld, 1990 ). There are approximately 100 times more ( + )RNA than ( -)RNA molecules in the infected cells . This relation varies, depending upon the cell line infected ( (Baltimore, 1966; Baltimore and Girard, 1966; Hewlett et al., 1977) . The single-stranded RNA found in cells as such is of positive polarity and it can be encapsidated in virions or serve as mRNA for the synthesis of viral proteins. In the encapsidated form, the virion RNA has a covalently bound protein (VPg) at the 5'-end of the molecule (Wu et al., 1978) . The RI found in cells consists of a complete (-)RNA molecule that is used as template to make copies of ( + )RNA. These are present as nascent RNA molecules (4-8 nascent (4-)RNA chains per RI), all of which contain a VPg molecule bound to the 5'-end. In fact, this molecule is present in all forms of RNA, positive or negative, that form part of RI or RF . It is well established that the enzyme required to elongate both (-) or (4-)RNA is 3D p°l Flanegan and Van Dyke, 1979) . This enzyme, however, is unable to initiate the synthesis of new RNA chains Semler et al., 1988; Richards and Ehrenfeld, 1990 ). 3D p°] is a polymerase that is able to copy RNA, using a primer and another RNA molecule as template (Richards and Ehrenfeld, 1990) . In fact, the first enzyme of this kind found in mammalian cells was described by Baltimore et al. (1963) in picornavirus-infected cells.

Apart from 3D p°~, the total number of proteins required for replication of the picornavirus genome has not yet been established. Virus-encoded proteins are necessary and cellular proteins may also be involved Semler et al., 1988; Richards and Ehrenfeld, 1990 ). In addition, viral (or cellular) proteins need to be synthesized continuously for participation in the synthesis of viral RNAs, because inhibition of protein synthesis in picornavirus-infected cells leads to almost immediate arrest of viral RNA synthesis (Fig. 21) .

Of the viral proteins implicated in the replication of poliovirus genomes, certainly 3D p°~ participates in the elongation process and VPg is also necessary Semler et al., 1988; Richards and Ehrenfeld, 1990) . It remains unclear if VPg acts as a primer to initiate the synthesis of new RNA chains or if VPg is added later after initiation has been accomplished (Takegami et al., 1983; Tobin et al., 1989) , and these two alternative possibilities are illustrated in Fig. 22 . According to model A, VPg, either as such or as the precursor 3AB, will, in the presence of UTP and a still unidentified protein, be uridylated to VPg-UMP and then further elongated to VPg-pUpUp (Takeda et al., 1986) . This substrate will be used as a primer to elongate the RNA chain (Takeda et al., 1986) . Both (-) and (+)RNA start with pUpUp at the 5'-end, and it is possible that VPg alone does not participate as such in the formation process, but as a precursor molecule 3AB . Thus, 3A would serve to anchor 3AB to the membrane in such a way that 3B would be "hanging" from the membrane and ready to be uridylated (Takegami et al., 1983; Wimmer et al., 1987) . Once 3AB-Up-Up is synthesized, or after the elongation process has commenced, 3C pr° will hydrolyze 3AB, releasing 3B from the membrane, while 3A would remain imbedded in the lipid bilayer . We have indicated that 3A is a firm candidate to modify membrane permeability and to lyse the infected cells (Lama and Carrasco, 1992a; Carrasco et al., 1993) . In model B of Fig. 22 , 3B (VPg) does not participate in a real initiation event, since the Y-OH group present in the hairpin can be used directly by 3D p°l to elongate the RNA chain. 3B (VPg) will bind somewhere during elongation in a reaction catalyzed by 3B itself with no requirement for nucleotide hydrolysis (Tobin et al., 1989) . Thus far, this model has not been confirmed by other laboratories.

Apart from the roles that 3D p°l, 3B (VPg), 3A and, indirectly, 3C p~° play in genome replication, other viral proteins are also implicated in this process although their functions are more obscure Semler et al., 1988; Richards and Ehrenfeld, 1990) . Genetic studies indicate that the functions of 2C, 2B and perhaps 2A pr° are also necessary for replication of viral RNA (Sarnow et al., 1990; Kirkegaard, 1992) . The most compelling evidence implicating 2C in RNA synthesis came from studies with the viral inhibitor guanidine (Caliguiri and Tamm, 1973) . This compound is a selective inhibitor of RNA synthesis in some picornaviruses (see Section 6.1), and elegant studies have proved that it acts on poliovirus protein 2C . In addition, poliovirus and mutations in 2C do not replicate the viral RNA (Li and Baltimore, 1988) . These mutants also have a defect in virion assembly, suggesting that 2C may also be involved in this late viral step (Li and Baltimore, 1990) . Sequence analysis of 2C suggested that it could be a GTPase because it possessed a Gly-rich region followed by the sequence GKS, a sequence present in some GTPases. Indeed, in vitro studies with the purified protein indicate that 2C has GTPase activity (Rodriguez and Carrasco, 1993) . A homology between picornavirus 2C and the potyvirus protein CI (Lain et al., 1989) has been suggested (Gorbalenya et al., 1990) . On this basis, it was claimed that 2C belongs to the RNA-helicase superfamily. However, no helicase activity has yet been demonstrated with isolated 2C (Rodriguez and Carrasco, 1993) , although this activity is present in 3D p°l (Cho et al., 1993) . In fact, we have suggested that 2C may represent a new class of small GTP-binding proteins and its role could be to direct the trafficking of RNA replicative complexes through the vesicular system (Rodriguez and Carrasco, 1993) . Clearly, more experiments are needed to clarify the activity of 2C in the replication of the poliovirus genomes. Protein 2B, either as such or as the precursor 2BC, is also required to replicate the picornavirus genome (Johnson and Sarnow, 1991) . Poliovirus with mutations in 2B is impaired in its ability to synthesize viral RNA, but there is no clue as to the exact role that this protein has in this process Sarnow et al., 1990; Kirkegaard, 1992) . Mutations in protein 2B cannot be complemented in trans, suggesting that only the actual 2B proteins made on a particular RNA are functional in the replication of that RNA (Johnson and Sarnow, 1991) . Indeed, this phenomenon very much complicates the possibility of using the picornavirus system as a model for the replication of any kind of RNA in cells or in cell-free systems (Kirkegaard, 1992) . Finally, 2A pr° may also participate in RNA replication. Apart from cleaving itself from P1, while the nascent polypeptide chain is being synthesized (Krausslich and , 2A pr° must have another role in viral growth. This idea is strongly supported by the fact that a poliovirus monster recently constructed, containing the P1 region separated from P2/P3 by the EMC 5' untranslated region, requires the presence of 2A or°, even though P2 proteins are now initiated on an internal AUG (Molla et al., 1992) .

The major evidence that host-factors are required for the replication of the picornavirus genome comes from cell-free systems (Semler et al., 1988; Richards and Ehrenfeld, 1990) . These systems are extremely useful for clarifying this process in molecular detail, but they are also prone to artifacts. Thus, a 67 kDa protein with uridylation activity has been isolated from infected cells that can replace the oligo(U) primer in the initiation of new poliovirus RNA chains (Dasgupta et al., 1980; Baron and Baltimore, 1982; Dasgupta, 1983) . This host factor was identified as the protein kinase that phosphorylates elF-2 (Morrow et al., 1985) . The role that such an activity may have in poliovirus-RNA replication has not been elucidated Semler et al., 1988; Richards and Ehrenfeld, 1990) , and the requirement of host factors for picornavirus RNA replication also requires further investigation.

The replication of poliovirus genomes takes place in close association with newly made membrane vesicles Semler et al., 1988; Richards and Ehrenfeld, 1990) . During poliovirus infection, the synthesis of membranes is profoundly modified, such that the cytoplasm is loaded with vesicles of various sizes that form a distinct perinuclear structure (Dales et al., 1965; Caliguiri and Tamm, 1970a, b; Mosser et al., 1972a) . This distinctive structure was termed viroplasm (Dales et al., 1965) . The synthesis of phospholipids increases after poliovirus infection to provide components for membrane generation (Mosser et al., 1972b) . The poliovirus replicative complexes are tightly associated with these newly made membranes (Girard and Baltimore, 1967; Tamm, 1969, 1970b; Bienz et al., 1990 Bienz et al., , 1992 Troxler et al., 1992) , although little is known about the origin of the membranous vesicles that appear in poliovirus-infected cells or the connection between these vesicles and other membrane compartments. The in vitro synthesis of poliovirus minus-or plus-stranded RNA has only been achieved using a crude membranous replication complex (Girard, 1969; Etchison and Ehrenfeld, 1981; Takeda et al., 1986) . Prevention of lipid synthesis results in an abrupt inhibition of viral RNA synthesis Carrasco, 1990, 1991) , suggesting that the replication of viral genomes requires continuous membrane formation (see Sections 6.10 and 6.12).

Elucidating the mechanisms used by poliovirus to replicate its genome is of interest for a number of reasons. For example, an understanding of the steps and proteins involved in this process sheds light on the relationship between the virus and the host cell (Koch and Koch, 1985; Wimmer et al., 1987; Semler et al., 1988; Richards and Ehrenfeld, 1990) . The complete synthesis of poliovirus in cell-free systems has already been achieved (Molla et al., 1991; Barton and Flanegan, 1993) , and this system should help to clarify the steps and components involved in poliovirus RNA replication. In addition, there is an interplay between the knowledge gained on poliovirus RNA synthesis and the inhibitors of this process, since a detailed understanding of the steps followed by poliovirus and other animal viruses in replicating their genomes is important for exploiting new approaches that interfere with the replication of these pathogens. Moreover, a better understanding of the mode of action of inhibitors of poliovirus RNA replication may help to elucidate this process.

Guanidine (Fig. 23) is a basic compound, positively charged at neutral pH, and is a natural constituent of animal serum. It is active against some members of the Picornaviridae and Togaviridae families (Friedman, 1970) and is, in many ways, a model inhibitor of poliovirus, despite the fact that it is one of the least potent agents against viral RNA synthesis (0.2-3 mM) (Tershak, 1974 (Tershak, , 1982 . Several guanidine derivatives have nevertheless been synthesized with more potent antiviral activity (Bucknall et al., 1973) . Soon after the discovery of guanidine as an antiviral agent (Rightsel et al., 1961) , poliovirus mutants resistant to or dependent on guanidine were described (Loddo et al., 1963; Nakano et al., 1963) . The picornaviruses inhibited by guanidine are poliovirus, rhinovirus and FMDV, whereas EMC virus is resistant to the compound (Caliguiri and Tamm, 1973; Tershak et al., 1982) . Guanidine has no adverse effects on cell growth at concentrations that block virus growth and has no direct effect on isolated virions, nor on virus attachment. When the inhibitor is added 3 hr after infection, it does not block translation, whereas the incorporation of [3H]uridine into poliovirus RNA is suppressed. If the compound is added after virus entry, the synthesis of viral proteins and of RNA are both prevented (Caliguiri and Tamm, 1973; Tershak et al., 1982) . Moreover, under low or moderate multiplicities of infection, guanidine largely prevents the shut-off of host protein synthesis induced by poliovirus, whereas at high multiplicity of infection, the kinetics of the early shut-off are similar in the absence or in the presence of inhibitory concentrations of guanidine (Lacal and Carrasco, 1983a) . For details on the initial studies on guanidine, the reader is referred to comprehensive reviews by Caliguiri and Tamm (1973) and Tershak et al. (1982) .

Early studies on the mode of action of guanidine indicated that this compound blocked the initiation of new rounds of viral RNA synthesis, but allowed completion of already initiated RNA strands Tamm, 1968a, b, 1973; Tershak et al., 1972) . It is not known with certainty whether guanidine is a selective inhibitor of the synthesis of plus-strand RNA, or blocks equally well the synthesis of plus-or minus-strand RNA. This indecision is due to the fact that it is difficult to block the synthesis of one strand of RNA without affecting the other. Careful kinetic analyses, using various concentrations of guanidine added at different times after infection, have not, to our knowledge, been carried out. Recently, the complete synthesis of infectious poliovirus has been accomplished in a cell-free system. In this system, poliovirus genomic RNA is added to a HeLa cell extract and after several hours of incubation, infectious poliovirus particles are made (Molla et ai., 1991) . Guanidine blocks the formation of these particles, indicating that at least under some conditions, the synthesis of plus-strand RNA and the initiation of RNA takes place in vitro (Molla et al., 1991) . The inhibition by guanidine ofpoliovirus or FMDV can be reversed by several amino acids, such as methionine, valine, leucine or threonine (Dinter and Bengston, 1964; Lwoff, 1965) , and by other agents, including methylpropanolamine, ethanolamine or choline Philipson et al., 1966) . This reversal very much depends on the host cell type analyzed. Transport of guanidine into HeLa cells involves at least two possible mechanisms (Nair, 1987b) that are regulated by the concentrations of cations in the medium (Nair, 1987a) . Some antiguanidine agents can block this transport, but this may not constitute the basis of their antiguanidine action; rather they could antagonize guanidine intracellularly by an unknown mechanism (Nair, 1987b).

As indicated earlier in this section, the feasibility of generating mutants allowed genetic mapping, which provided information on the viral protein that was the target of guanidine action (Anderson Sillman et al., 1984; Pincus et al., 1987) . Initial studies suggested that capsid proteins, proteases or even VPg were the targets of guanidine. Very detailed analyses of both guanidine-resistant and guanidine-dependent mutants of poliovirus indicate that they map in protein 2C Baltera and Tershak, 1989) , and similar conclusions were reached for FMDV guanidine mutants (Saunders and King, 1982) . Analyses of the polypeptides of FMDV or poliovirus guanidine-resistant mutants suggested that protein 2C was responsible for this trait. Transfection of COS cells, with plasmids containing eDNA fragments corresponding to poliovirus protein 2C from guanidine-resistant or guanidine-dependent mutants, and with plasmids containing infectious clones of wild-type poliovirus, gave rise to both types of mutant virus . Sequence analyses indicated that mutations in amino acid residue 179 of protein 2C conferred resistance to high guanidine concentrations, whereas changes in residue 187, combined with mutations at position 142, 225 or 227, generate guanidine-dependence . Modifications in residue 164 or 187 of poliovirus protein 2C, combined with mutations at residues 223 or 295 and 309, could also confer guanidine resistance (Baltera and Tershak, 1989) . Finally, the frequency of reversion of guanidine-dependent mutants of poliovirus to the resistance phenotype has established a mutation frequency of about 6 x 10 -4, a number consistent with the genetic variability of RNA-containing viruses (Domingo, 1989 (Domingo, , 1992 Holland et al., 1992; Drake, 1993 ).

An updated view of the mode of action of guanidine could be as follows: guanidine acts on protein 2C, perhaps inhibiting the interaction of this protein with membranes . Protein 2C is a GTPase that may be involved in trafficking the viral RNA replication complexes through the vesicular system (Rodriguez and Carrasco, 1993) . This traffic is necessary for poliovirus, but not EMC virus, genome replication (Irurzun et al., 1992) . Thus, guanidine blocks the activity of 2C, the traffic of replication complexes and, hence, the initiation of viral RNA synthesis. This mode of action may also explain why guanidine affects other steps, such as the assembly of new virions that also takes place in close association with the newly-formed membrane vesicles (Koch and Koch, 1985) .

Guanidine and some of its derivatives have been used in several pharmacological studies (Davidoff, 1973) . This compound provokes an enhanced nerve excitability after injection into frogs and may play a role in the pathogenesis of uremic poisoning. Although the serum levels of guanidine are similar in control and uremic patients, the excretion of methylguanidine is higher in patients with renal failure (Stein and Micklus, 1973) . These findings, together with the fact that high concentrations (~ 1 mM) of guanidine are required for efficient blockage of poliovirus in vitro, indicate that guanidine cannot be used therapeutically. Guanidine is ineffective for the treatment of poliovirus infection in animals, because of the rapid secretion of this compound and the rapid development of guanidine-resistant viruses in animals (Caliguiri and Tamm, 1973) . In fact, administration of guanidine alone to mice infected with echovirus 9 or coxsackie virus A9 did not protect them. A partial response was observed after combination of 2-(~t-hydroxybenzyl)benzimidazole (HBB) and guanidine, as indicated by several days' delay, before death (Eggers, 1976) .

Thiosemicarbazones (Fig. 24) have been used as therapeutic agents for the treatment of tuberculosis and as antiviral agents against several animal viruses (Levinson, 1975; Pfau, 1982) . Isatin-fl-thiosemicarbazone inhibits vaccinia virus and rhinoviruses (Katz and Feliz, 1977; Pennington, 1977) , but has no effect against poliovirus (Pearson and Zimmerman, 1969) . The activity of methisazone (N-methyl-isatin-fl-thiosemicarbazone) against several picornaviruses was reported (Bauer et al., 1970) , and various species of viruses, including poliovirus, were directly inactivated upon contact with methisazone in the presence of CuSO4 (Fox et aL, 1977) . 1-Methyl-4',4'-dibutylisatin-/~-thiosemicarbazone (busatin) blocks poliovirus (O'Sullivan and Sadler, 1961; Pearson and Zimmerman, 1969) , interfering with the synthesis of viral RNA when added at any time during infection (Pearson and Zimmerman, 1969) . In contrast with the action of HBB (see Section 6.3) or guanidine, busatin blocks poliovirus RNA synthesis in cell-free systems (Pearson and Zimmerman, 1969) , and in this respect shows similarity to gliotoxin (Rodriguez and Carrasco, 1992) .

Some derivatives of thiosemicarbazones, the 3-substituted triazinoindoles (SKF 21687, SKF 30097, SKF 36800, SKF 40491) (Fig. 24) , are broad-spectrum antipicornavirus agents that block several rhinovirus strains (Matsumoto et al., 1972; Pfau, 1982) . SKF 40491 has antirhinovirus activity when administered intranasally to gibbons, but not when used orally (Pinto et aL, 1972) . SKF 21687 and SKF 30097 had no effect on rhinovirus infection in humans (Togo et ai., 1973b) .

Several benzimidazole derivatives (Fig. 25) show activity against a number of animal viruses (Caliguiri and Tamm, 1973; Tamm and Sehgal, 1978; Eggers, 1982 Eggers, , 1985 . For example, HBB is a selective inhibitor of picornavirus replication that does not inactivate virion particles, but blocks the replication of viral RNA without affecting the synthesis of cellular RNA (Eggers and Tamm, 1962; Singh et al., 1963) . The structure-activity relationships in HBB derivatives are fairly well documented (Caliguiri and Tamm, 1973; Tamm and Sehgal, 1978; Eggers, 1982 Eggers, , 1985 , showing that the benzo group and the hydroxybenzyl residue at position 2 are critical for activity. Some HBB derivatives have been reported to be more active than HBB itself (Caliguiri and Tamm, 1973; Tamm and Sehgal, 1978; Eggers, 1982 Eggers, , 1985 .

HBB is not a broad-spectrum antipicornavirus agent, being active against poliovirus and some coxsackie viruses, but devoid of activity against rhinoviruses, FMDV or hepatitis A virus (Siegl and Eggers, 1982) . The concentration of HBB required for activity against poliovirus is similar to that of guanidine, whereas HBB is about 10-fold more potent than guanidine against coxsackie virus. The action of HBB on poliovirus, as found for that of guanidine and flavones, is reversible. Upon withdrawal of these compounds, virus growth resumes (Caliguiri and Tamm, 1973; Tamm and Sehgal, 1978; Eggers, 1982 Eggers, , 1985 .

Studies on the mode of action of HBB indicate that it has no effect on early steps of infection, such as virus entry and uncoating (Eggers and Tamm, 1962) . Addition of the compound after poliovirus entry prevents the appearance of viral proteins, because viral RNA replication is blocked. Thus, the incorporation of labelled uridine into viral RNA and the formation of poliovirus-specific RNA, as analyzed by dot-blot hybridization, are inhibited by HBB (Eggers and Tamm, 1963a; Gonz/tlez et al., 1990b) . This compound has no effect on in vitro synthesis of viral RNA, and it is still not known if the initiation or elongation steps are selectively blocked by this agent in vivo.

A number of picornavirus mutants that are resistant to or dependent on HBB have been isolated, but they are not cross-resistant to guanidine (Eggers and Tamm, 1963b) . The dependent mutants require HBB for the replication of viral RNA, and HBB can be replaced by guanidine in this respect (Eggers and Tamm, 1963b) . Mutant HBB-dependent viruses back-mutate to the sensitive phenotype with high frequency (,~ 10 -4) Caliguiri and Tamm, 1973) , this figure being similar to the mutation rate of picornaviruses for other traits investigated (de la Torre et al., 1990; Domingo, 1992 

HBB and guanidine act synergistically (Singh et al., 1963) , suggesting that the exact target inhibited by each compound is different, although they could act on the same viral protein.

Investigations of the antiviral effects of HBB in mice and monkeys have been carried out (Caliguiri and Tamm, 1973; Eggers, 1982) . Although early experiments yielded only marginal effects of HBB on virus-infected animals, newborn mice inoculated with echovirus 9 or coxsackie virus A9 were protected by combined treatment with HBB and guanidine (Eggers, 1976) . HBB alone protected coxsackie virus-infected animals, but not those infected with echovirus (Eggers, 1976 ).

Enviroxime (2-amino-l-(isopropylsulfonyl)-6-benzimidazole phenyl ketone oxime) (Fig. 25) is a benzimidazole derivative with high activity against rhinoviruses (Delong and Reed, 1980) , and several human trials using enviroxime as a prophylactic agent against the common cold produced by rhinoviruses have been reported (Hayden and Gwaltney, 1982; Levandowski et al., 1982; Phillpots et al., 1983b) . No clear benefit was observed (Levandowski et al., 1982) , although incorporation of enviroxime into liposomes results in improved drug delivery to the lungs of mice (Wyde et al., 1988) . Studies on the mode of action of this drug have been retarded because the compound is not freely available from Eli Lilly Research Laboratories for research studies.

Flavonoids are ubiquitous compounds in the plant kingdom (Harborne, 1988) . The search for antiviral agents in plant extracts led to the discovery of 3-methyl quercetin (3-MQ) from the African medicinal plant Euphorbia grantii (Van Hoof et al., 1984; Vlietinck et al., 1986) and Ro 09-0179 (Fig. 26) from the Chinese medicinal herb Agastache rugosa (Ishitsuka et al., 1982b) . Analysis of the mode of action of 3-MQ indicated that it was a selective inhibitor of poliovirus RNA synthesis. At a concentration of 5 #g/mE 3-MQ inhibited [3H]uridine incorporation in poliovirus-infected cells by 90%, whereas at 20/~g/mL, 3-MQ had no effect on cellular transcription (Castrillo et al., 1986) . Analysis of the viral RNAs synthesized in the presence of this flavone showed that production of single-stranded RNA was totally blocked, whereas the synthesis of dsRNA was still detectable (Castrillo and Carrasco, 1987a ). Thus, 3-MQ behaves like Ro 09-0179 in blocking the synthesis of both plus-and minus-RNA when added from the inception of infection, because there is not enough genomic RNA to serve as a template to give rise to significant amounts of negative-stranded RNA that can be detected by hybridization (Lopez Pila et al., 1989; Gonz/dez et al., 1990b) . Therefore, in order to test if these flavones are selective, they must be added later during infection, when sufficient amounts of plus-and minus-stranded RNA are being made. Addition of Ro 09-0179 late in infection affects viral RNA synthesis in a way that suggests a preferential inhibition of poliovirus genomic RNA (Gonzfilez et al., 1990b) . However, a detailed understanding of the molecular mode of action of both 3-MQ and Ro 09-0179 is still lacking, because we do not know yet which viral protein is their target. It is also not known if the initiation, elongation or both these steps of viral RNA synthesis are blocked by these compounds. Moreover, we have recently found that some flavones may block early steps of poliovirus growth, suggesting that some flavones could directly interact with viral particles and block uncoating (A. Irurzun and L. Carrasco, unpublished results) .

3-MQ and Ro 09-0179 inhibit poliovirus replication at concentrations 100-fold, or 1000-fold, lower than those shown for HBB or guanidine, respectively (Gonzfilez et al., 1990b) . Ro 09-0179 selectively blocks viral RNA synthesis in poliovirus-infected HeLa cells more strongly than 3-MQ, and these findings make Ro 09-0179 a valuable compound for obtaining insight into the molecular mechanisms of poliovirus RNA replication.

Quercetin and morin are effective against mengovirus-induced encephalitis in mice when given orally (Veckenstedt et al., 1978) , but these two flavonoids do not show selectivity for inhibition of poliovirus in cultured cells (Castrillo et al., 1986; Gonz/dez et al., 1990b) . The diacyl derivative of Ro 09-0179 is absorbed better than the parent compound in the intestinal tract, making it effective against lethal coxsackie B1 infections in mice (Ishitsuka et al., 1982b) .

Gliotoxin (Fig. 27) is a fungal metabolite produced by various species of Trichoderma, Aspergillus and Penicillium, and it possesses different biological activities, including antiviral effects (Suhadolnik, 1967a; Miller et al., 1968; McDougall, 1969) . Gliotoxin and a number of related compounds, derived either from natural sources or by chemical synthesis, are the most potent agents that interfere with poliovirus RNA synthesis (Miller et al., 1968; Trown and Bilello, 1972) . The presence of a reducing agent such as dithiothreitol prevents the inhibition of poliovirus RNA synthesis in intact cells (Trown and Bilello, 1972) , suggesting that blockade by gliotoxin involves the formation of disulfide bridges between gliotoxin and essential sulfhydryl groups on the viral polymerase. The presence of dithiothreitol in cell-free systems partially lowered the gliotoxin inhibitory potential, in agreement with the idea that reducing the sulfhydryl groups abolishes the activity of gliotoxin (Trown and Bilello, 1972; Rodriguez and Carrasco, 1992) .

The mode of action of gliotoxin against poliovirus has been analyzed in detail (Rodriguez and Carrasco, 1992) . This fungal metabolite irreversibly inhibits the appearance of poliovirus proteins when present from the beginning of infection, but has no effect on viral translation when added later. This toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium (Miller et al., 1968; Rodriguez and Carrasco, 1992) . Analysis of the synthesis of poliovirus plus-or minus-stranded RNA in the presence of gliotoxin indicated that this compound effectively hampered both processes (Rodriguez and Carrasco, 1992) , a result which contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as flavones, that selectively block plus-strand RNA synthesis and suggests that the target of gliotoxin may differ from the target of flavones. Gliotoxin was a potent inhibitor ofpoliovirus RNA synthesis in cell-free systems using membranous crude replication complexes, a reaction that is not blocked by guanidine, HBB or Ro 09-0179. Moreover, an in vitro assay, using the purified poliovirus polymerase 3D p°t, was efficiently inhibited by gliotoxin, the first description of an inhibitor of this viral enzyme (Rodriguez and Carrasco, 1992) .

Gliotoxin is rather toxic for animals, the LDs0 for mice being 7.8 mg/mL intravenously and 32 mg/mL intraperitoneally (Larin et al., 1965) . Despite this toxicity, gliotoxin exerts antiviral effects in monkeys inoculated with large doses of poliovirus (Larin et al., 1965) .

Antipicornavirus activities have also been described for other compounds related to gliotoxin.

These include aranotin, acetylaranotin and apoaranotin (Fig. 27) , all isolated from the culture medium of the fungi Arachniotus aureus and Aspergillus terreus (Trown, 1968) , and sirodesmins (Fig. 27) , isolated from the fungus Sirodesmium dioersum. Sirodesmins A, B and C differ in the number of sulfur atoms in the bridge, being two, three or four, respectively. Several hyalodendrins (Fig. 27) , which also differ in the number of sulfur atoms in the bridge, were isolated from the fungi Hyalodendrin species and Penicillium turbatum (Michel et al., 1974) . These compounds inhibited poliovirus, rhinovirus and coxsackie virus multiplication (Trown, 1968; Michel et al., 1974) . The synthetic compound N,N'-dimethylepidithiapiperazinedione (Fig. 27) was also a very potent inhibitor of coxsackie virus RNA synthesis (Trown, 1968 ).

A natural compound, aldgamycin E (Fig. 28) , a macrolide antibiotic produced by Streptomyces lavandulae, inhibits the growth of poliovirus, but not of other animal viruses tested (Gonz~lez and Carrasco, unpublished results), A blockade of viral translation takes place when the antibiotic is present from the inception of infection, but no effect on poliovirus protein synthesis was observed when this agent was added later, suggesting that protein synthesis was not the primary target for antibiotic action. Indeed, poliovirus RNA synthesis was profoundly depressed soon after the addition of aldgamycin E. Other macrolide antibiotics, such as chalcomycin, josamycin and venturicidin, also blocked poliovirus RNA synthesis, indicating that macrolide antibiotics constitute a promising source of new antiviral agents (Gonz/dez and Carrasco, unpublished results). 

Dipyridamole (Fig. 26) is a synthetic compound which is a coronary vasodilator. Dipyridamole and several of its derivatives are active against certain DNA and RNA viruses in cell culture, including mengovirus (Kuwata et al., 1977; Tonew et al., 1982) . The synthesis of mengovirus RNA was completely blocked by dipyridamole under conditions where the transport of uridine was not affected, i.e. by precharging the cells with labelled uridine (Kuwata et al., 1977; Tonew et al., 1982) , but more detailed analyses, using hybridization with specific probes, are necessary because dipyridamole also affects nucleoside transport.

As indicated in Section 6.1, the action of guanidine can be antagonized by a number of amino acids, and on this basis, it was suggested that certain analogues of these amino acids might also show antipoliovirus activity . D-Penicillamine (Fig. 26) , a valine analogue, inhibits poliovirus growth reversibly and blocks the synthesis of poliovirus RNA at drug concentrations of 0.1-0.5 mg/mL . The relative concentrations of single-stranded and double-stranded poliovirus RNA did not vary in the presence of this compound. Certainly, D-penicillamine is not a potent poliovirus inhibitor, because at a drug concentration of 1 raM, total blockade of viral DNA synthesis was not achieved (Merryman et al., 1974) . The inhibitory effect of D-penicillamine is reversible and can be antagonized by several amino acids.

One of the most potent inhibitors of lipid biosynthesis in prokaryotic and eukaryotic cells is cerulenin (Fig. 28) , an antibiotic produced by Cephalosporium caerulens (Bachrach et al., 1973) . It blocks fatty-acid synthetase at the level of fl-ketoacyl thioester synthetase (condensing enzyme) (Armstrong et al., 1972; Butterworth and Rueckert, 1972; Bachrach et al., 1973) . If present from the initiation of poliovirus infection, there is a potent inhibition of poliovirus protein synthesis, but it has no effect on viral translation or on the proteolytic processing of the viral proteins, if added late (Guinea and Carrasco, 1990) . Myristoylation of the viral protein VP4 or its precursors is not blocked by cerulenin. Cerulenin inhibits very quickly the replication of poliovirus RNA when added at any time during infection (Guinea and Carrasco, 1990) , whereas it has no effect on cellular transcription. This antibiotic depresses the incorporation of labelled glycerol into phospholipids, and this inhibition parallels the blockade of viral RNA replication. The synthesis of poliovirus plus-stranded RNA synthesis is hampered (Guinea and Carrasco, 1990 ). Cerulenin does not have a direct inhibitory effect on poliovirus translation, indicating that the synthesis of phospholipids is continuously required for the synthesis of plus-stranded poliovirus RNA. Hence, there is some link between membrane traffic and poliovirus RNA replication.

This antibiotic is also an inhibitor of some enveloped RNA-containing viruses (Schlesinger and Malfer, 1982; Ikuta and Luftig, 1986; Pal et al., 1988) . Thus, the replication of retroviruses, including HIV type 1, is blocked by cerulenin (Pal et al., 1988) . It was proposed that the processing of structural proteins and the acylation of some glycoproteins could be targets for cerulenin in its inhibition of viral growth (Schlesinger and Malfer, 1982; Ikuta and Luftig, 1986; Pal et al., 1988) . The inhibition of phospholipid synthesis by cerulenin also impairs the synthesis of nucleic acids in other viruses, such as EMC virus, SFV and VSV (Guinea and Carrasco, 1990; . In fact, the synthesis of nucleic acids, not only in togaviruses, but in many other animal viruses, is closely connected to the synthesis of new membranous structures. If the synthesis of these membrane structures requires de novo synthesis of phospholipids, it seems plausible that inhibition of lipid synthesis would certainly lead to inhibition of viral nucleic acid synthesis .

Other compounds that block phospholipid synthesis include TOFA and hydroxyacetic acid (Fig. 28) , which act in a similar manner as cerulenin (Guinea and Carrasco, unpublished results) . Clofibrate, a compound that potentiates the oxidation of fatty acids, pentenoic acid, an inhibitor of fatty acid oxidation, hemicholinium, an inhibitor of choline incorporation into phosphatidylcholine, and compactine, an inhibitor of cholesterol synthesis, all had no effect on poliovirus. On the other hand, agaric acid, an inhibitor of fatty acid synthesis, sodium palmoxirate, another agent that blocks fatty acid oxidation, and 3-morpholine-l-propanol, were all too toxic in control cells to allow assay of their effects on poliovirus-infected cells (Guinea and Carrasco, unpublished results) . TOFA and hydroxycitrate, two known inhibitors of lipid synthesis, blocked the appearance of poliovirus proteins when present from early stages of infection. Further analyses on the effects of these two compounds on poliovirus replication indicated that they had no action on poliovirus protein synthesis if added late during infection, whereas they potently depressed poliovirus RNA synthesis (Guinea and Carrasco, unpublished results) . Furthermore, both compounds also had an inhibitory effect on phospholipid synthesis in poliovirus-infected cells.

These results indicate that agents that interfere with phospholipid biosynthesis, such as cerulenin, TOFA and hydroxycitrate, hamper the replication of poliovirus RNA. Thus, the mode of action of these agents differs from that of the well-known poliovirus RNA inhibitor guanidine, because guanidine has no effect on lipid synthesis.

Membranes are continuously in motion through several compartments in eukaryotic cells (Morr6 and Ovtracht, 1977; Rotbman and Orci, 1992; Lippincott-Schwartz, 1993) . Brefeldin A (BFA) (Fig. 28) , a fungal antibiotic isolated from Penicillium brefeldianum, has antiviral properties and blocks protein secretion and vesicular traffic (Hunziker et al., 1992; Klausner et al., 1992) . It induces a rapid disaggregation of the Golgi complex, causing redistribution into the endoplasmic reticulum (Lippincott-Schwartz et al., 1989 Alcalde et al., 1991) . Within seconds of BFA treatment, a peripheral protein (110 kDa) of the Golgi apparatus is released in the cytoplasm and a Golgi membrane enzyme that catalyzes exchange of guanine nucleotide onto ARF is inhibited Helms and Rothman, 1992) . Since transport of proteins into post-Golgi compartments in the cell is potently blocked by BFA , so too is the delivery of VSV G glycoprotein to the plasma membrane (Misumi et al., 1986; Fujiwara et al., 1988; Doms et aL, 1989; Oda et al., 1990) . In addition to VSV, an increasing number of animal viruses are reported to be affected by this inhibitor (Ulmer and Palade, 1990; Chen et al., 1991; Cheung et al., 1991; Whealy et al., 1991) . BFA inhibits the processing and transport of glycoproteins that are present in all these viruses. In this manner, enveloped viruses that mature from the plasma membrane or mature intracellularly are inhibited by this macrolide antibiotic (Ulmer and Palade, 1990; Chen et al., 1991; Cheung et al., 1991; Whealy et al., 1991) .

BFA strongly inhibits poliovirus replication (Irurzun et al., 1992; Maynell et al., 1992) , even though this virus lacks a lipid envelope and does not encode any glycoproteins (Urzainqui and Carrasco, 1988) . Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins, but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2A pr° (Irurzun et al., 1992) . The presence of BFA at later times has no effect on poliovirus protein synthesis, so that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labelled uridine into poliovirus RNA (Irurzun et al., 1992; Maynell et al., 1992) . Both plus-and minus-stranded RNA synthesis are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times (Irurzun et al., 1992) .

The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species (Irurzun et al., 1992) . Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas EMC virus is resistant. A negative-stranded RNA virus, such as vesicular stomatitis, is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant (Irurzun et al., 1992) . It follows that even viruses that do not contain a lipid envelope and do not synthesize glycoproteins, may require newly made membranes to allow the synthesis of new genomes (Irurzun et al., 1992; Maynell et al., 1992) . If so, the poliovirus proteins, once synthesized, will bind to membranes in the rough endoplasmic reticulum and then migrate through the Golgi complex, following the same route used by glycoproteins (Fig. 29) . Once the poliovirus proteins reach the trans-Golgi network, they will be assigned to the specialized structures formed by the membranous vesicles and the RNA replication complexes (see Section 5.2). The finding that BFA immediately blocks poliovirus RNA synthesis without affecting translation suggests that RNA synthesis takes place in association with newly made vesicles and that preexisting vesicles do not support the formation of new genomes. These findings will help to clarify the action of a number of antiviral agents that interfere with the replication of genomes without affecting the viral polymerase itself. In addition, they can be exploited to devise more selective antiviral agents because, at least theoretically, we could interfere with the action of the viral proteins involved in the coupling between the membranous vesicles and the RNA replication complexes.

Addition of some fatty acids to cells in culture increases lipid synthesis (Cowen and Heydrick, 1972; Burkhardt, 1991) . Conversely, unsaturated fatty acids, such as oleic acid, increase membrane fluidity and diminish the infectivity and hemolytic activity of enveloped animal virus particles. No such effects have been described for non-lipid-enveloped animal virus particles (Kohn et al., 1980; Sola et al., 1986a) . Oleic acid (Fig. 28 ) blocks the synthesis of poliovirus proteins at concentrations that do not affect translation in uninfected cells . This inhibition is due to a blockade of viral RNA synthesis, most probably as a consequence of modifications in the membranes connected with the RNA replication complexes. Addition of oleic acid, but not other fatty acids, from the beginning of poliovirus infection, specifically inhibits the appearance of virus polypeptides at concentrations that do not affect translation in mock-infected HeLa cells. This inhibition is most probably due to the blockade of viral RNA synthesis, because oleic acid interferes with the synthesis of poliovirus RNA both in vivo and in cell-free systems . Oleic acid increased the synthesis of phosphatidylcholine and most neutral lipids. Membranes made in poliovirus-infected cells in the presence of oleic acid differ in their buoyant density from control membranes. The incorporation of oleic acid into membranes leads to increased membrane fluidity and decreased buoyant density, making these membranes nonfunctional for RNA replication . Therefore, not only is the synthesis of phospholipids required for viral RNA synthesis (Guinea and Carrasco, 1990) , but the proper structure of the newly made membranes is also important to permit replication of poliovirus genomes.

Interferons are cellular proteins, synthesized in response to several inducers (Stringfellow, 1981; Sehgal et al., 1982) , that possess antiviral activities (De Maeyer et al., 1981; Staeheli, 1990; Kerr and Stark, 1992) . The interaction of the interferon molecule with the appropriate receptor on the cell surface induces the synthesis of a number of proteins, thus establishing the antiviral state (Kerr and Stark, 1992; McNair and Kerr, 1992) . The precise action of these proteins against the replication of animal viruses remains largely unknown (Taylor and Grossberg, 1990) .

In the case of picornaviruses, it is well established that treatment of susceptible cells with different interferon species profoundly blocks viral growth (see review by Mufioz and Carrasco, 1987) . Treatment of cells with human interferon, followed by subsequent infection with poliovirus, does not prevent inhibition of host protein synthesis, indicating that translation of the input viral RNA takes place (Mufioz and Carrasco, 1983) . Isolation of RNAs from poliovirus-infected cells treated with interferon indicated that the ribosomal RNAs remained intact and that the cellular mRNAs were translatable, so that degradation of cellular RNAs is not the basis of interferon action (Mufioz et aL, 1983 ). The precise step inhibited in picornavirus growth in cells treated with interferon remains largely unexplored, despite the large number of publications investigating this effect in cell-free systems and a good knowledge of the particular enzymes induced by interferon in cells (Mufioz and Carrasco, 1987) . It is quite probable that the synthesis of picornaviral RNA is the step blocked in interferon-treated cells (Mufioz and Carrasco, 1983; Mufioz et al., 1983) , but it is assumed by many groups that interferons primarily block viral translation with this effect being mediated by the PPP(A2'p5'A)nA (2'-5' A) synthetase and/or the P1 protein kinase upon activation by dsRNA (Taylor and Grossberg, 1990; Kerr and Stark, 1992; Zhou et al., 1993) . It is difficult to establish whether interferon primarily blocks transcription or translation, not only for picornaviruses, but also for other RNA viruses, because interference with viral RNA synthesis usually leads to a reduction in viral protein synthesis and vice versa. Nevertheless, in the case of poliovirus, translation of the input RNA is not impaired in interferon-treated cells (Mufioz and Carrasco, 1983) . Moreover, human cells treated with interferon and doubly-infected with poliovirus and reovirus synthesize reovirus proteins, even though poliovirus replication is impaired (Mufioz et al., 1985a; Mufioz and Carrasco, 1987) . These findings indicate that there is no indiscriminate inhibition of viral proteins in interferon-teated cells and infected with poliovirus, because these cells make reovirus proteins at control levels, even though the antiviral state against poliovirus exists in the same cells.

The mechanism underlying the blockade of picornaviruses by interferon is also unclear. If interferon blocks viral RNA synthesis, how is this accomplished? The possibility that interferon blocks translation of EMC virus or mengovirus through activation of the 2'-5' A synthetase and/or the P1 protein kinase has been investigated (Verhaegen-Lewalle et al., 1982; Rice et al., 1985; Kumar et al., 1988; Lewis, 1988) . Various reports show no correlation between the activation of these enzymes and the establishment of an antiviral state, not only in picornavirus-infected cells Verhaegen-LewaUe et al., 1982; Kumar et al., 1988; Lewis, 1988) , but also in other viral systems (Mufioz and Carrasco, 1987; Taylor and Grossberg, 1990) . Even in the case of reoviruses that contain dsRNA as genome, there is no inhibition of viral protein synthesis in HeLa cells even though the 2'-5' A synthetase and the P1 protein kinase have been induced (Feduchi et al., 1988) . Transfection of the 2'-5' A synthetase or the P1 protein kinase inhibit virus growth (Chebath et al., 1987; Coccia et al., 1990; Meurs et al., 1992; Lee and Esteban, 1993) , but it remains to be determined if this inhibition is related to the mode of action of interferon, because cells transfected with the 2'-5' A synthetase gene support the growth of VSV (Chebath et al., 1987; Coccia et al., 1990) . Furthermore, vaccinia virus, which is resistant to interferon blockade, is inhibited in cells transfected by the P1 protein kinase gene (Meurs et al., 1992; Lee and Esteban, 1993) . The intracellular location of the 2'-5' A synthetase and the role that this enzyme plays in cellular metabolism deserve further investigation.

Cell-free systems from interferon-treated cells synthesize a series of oligo(A) compounds in the presence of ATP and dsRNA (Williams et al., 1978 (Williams et al., , 1979 Kerr et al., 1982; Kerr and Stark, 1992) . These oligo(A)s characteristically have phosphodiester bonds that are 2'-5' instead of the usual 3'-5' bonds. They are collectively referred to as 2'-5' A ( Fig. 30) and are synthesized in minute amounts both in normal cells and in interferon-treated cells after virus infection Laurence et al., 1984; Kerr and Stark, 1992) , but they can be synthesized in higher amounts in extracts or by using the purified 2'-5' A synthetase, or even chemically (Haugh et al., 1983; Kerr and Stark, 1992) . The exact role that the 2'-5' A plays in the antiviral action of interferon remains obscure, because there is no correlation between the antiviral action of interferon and the presence of 2'-5' A synthetase in cultured cells (Jacobsen et al., 1983; Lewis, 1988; Taylor and Grossberg, 1990) . Apart from the role that oligo(A) could play in the action of interferon against animal viruses, direct addition of pppA2'p5'A to the cell medium prevents virus infection (Alarcrn et al., 1984a) . pppA2'p5'A is active against several animal viruses, including EMC virus and poliovirus, at concentrations between 0.05 and 0.5 mM. This compound does not inhibit translation; rather it blocks an early step during infection of VSV. The mode of action against picornaviruses remains undetermined (Alarc6n et al., 1984a) .

Since 2'-5' A is a charged molecule that does not enter easily into cells, the 2'-5' A core, or a number of derivatives, have been used to inhibit translation and to block virus infection in intact cells (Imai et al., 1982; Eppstein et al., 1982; Haugh et al., 1983; Sawai et al., 1983; Goswami et al., 1984; Eppstein et al., 1986) . The xyloadenosine analogue of 2'-5' A that is dephosphorylated at the Y-terminus is much more stable and more active than the 2'-5' A core. Phosphorothioate analogues of 2'-5' A interfere with the activation of RNase L in HeLa cells, perhaps by competition with authentic 2'-5' A (Charachon et al., 1990) . Cordycepin or 3-methyl analogues of 2'-5' A have also been synthesized. However, although these analogues had antiviral effects against several DNA-containing viruses in cultured cells, their precise mode of action against picornaviruses is not known.

Double-stranded polynucleotide complexes mediate interferon induction, thereby exerting antiviral effects (Shugar, 1974; Sehgal et al., 1982; Shannon, 1984; Streissle et al., 1985) . Apart from this action, polynucleotides themselves may have antiviral activity (Shugar, 1974; Shannon, 1984; Stebbing, 1984; Streissle et al., 1985) . Thus, protection of mice against EMC virus has been observed with poly (I), tRNA or poly (C) (Stebbing et al., 1976 (Stebbing et al., , 1977 . However, the antiviral action of these polynucleotides does not seem to be mediated by interferon induction and subsequent stimulation of the immune system, because protection was also achieved in immunosuppressed mice or athymic mice (Stebbing et al., 1976) .

Other approaches used to block virus infection involve antisense RNA and ribozymes (James, 1991) . Antisense oligonucleotides or oligonucleotide derivatives possess good activities against a variety of animal viruses (Stephenson and Zamecnik, 1978; Goodchild et al., 1988; Leonetti et al., 1990) . Particularly effective are poly (L-lysine) conjugates (Degols et al., 1989) and phosphorothioate oligodeoxynucleotides, because they more readily enter cells and are more stable (Leiter et al., 1990; Agrawal et al., 1992) . Phosphorodithioate DNA oligomers are powerful inhibitors of HIV-1 reverse transcriptase (Marshall and Caruthers, 1993) . To my knowledge, this approach has not been used to block picornaviruses.

Until the 1980s the synthesis of nucleoside analogues was largely directed towards inhibition of members of the Herpesviridae family. This led to the development of a most effective antiviral agent, acycloguanosine (Schaeffer et al., 1978; Elion, 1989) . Research on nucleoside analogues in the last decade has concentrated on finding selective inhibitors of HIV. Some nucleotide analogues possess a broad spectrum of action and some of them also block members of the Pieornaviridae family. However, this is not a general rule because certain of these analogues, such as (S)DHPA or aristeromycin, have no effect against poliovirus (De Clercq et al., 1978 .

Although some nucleoside analogues may have a single viral enzyme as their site of action, some of them may affect a variety of cellular and viral enzymes (Carrasco and Vhzquez, 1984) . This is particularly true for adenosine (ATP) or guanosine (GTP) analogues, since these nucleosides and nucleotides participate in a variety of functions. Therefore, it can be anticipated that the action of some nucleoside analogues will be rather complex (Carrasco and V~zquez, 1984) . To our knowledge, the mode of action of none of the nucleoside analogues active against picornaviruses has been analyzed in detail, although some of them, such as ribavirin (l,~-D-ribofuranosyl-l,2,4triazole-3-carboxamide), show interesting activity.

Pyrazofurin (3(fl-o-dbofuranosyl)-4-hydroxyrazole-5-carboxamide) (Fig. 31) , originally called pyrazomycin, was isolated from the fermentation broth of Streptomyces candidus. This antibiotic displayed wide-spectrum antiviral activity with a very good antiviral index. It is active against rhinovirus, and in this respect is more potent than ribavirin ). An excess of uridine in the culture medium abolished the antiviral effects, suggesting that the drug blocked steps in the metabolism of nucleotides (Gerzon et al., 1971) . Pyrazofurin-5'-phosphate is an antagonist of uridine metabolism and blocks orotidylic deearboxylase (Shugar, 1974; Gutowski et al., 1975) , but it also inhibits 5-amino-imidazole-4-carboxamide-l-fl-D-ribofuranosyl-5"-monophosphate (AICAR) formyltransferase, an enzyme involved in purine nucleotide biosynthesis. Inhibition of IMP-dehydrogenase activity by related compounds such as ribavirin has also been reported (Streeter et al., 1973; Robins et al., 1985; Yamada et al., 1988) . Ribavirin (Fig. 31) was chemically synthesized and proved to be also a wide-spectrum antiviral agent (Witkowski et al., 1972; Streeter et al., 1973; Hahn, 1979 ) that shows activity against HIV . This compound has good antiviral activity in several animal models and in humans (Hahn, 1979; Hayden, 1985; Fernandez et al., 1986; Patterson and Fernandez Larsson, 1990) and has been licensed in the USA for the treatment of respiratory syncytial virus infections. Ribavirin shows antiviral activity at concentrations well below those that cause cytotoxicity (Hahn, 1979; Sidwell et al., 1979; Smith and Kirkpatrick, 1980) , although the mode of action of this interesting antiviral agent remains obscure. The idea that ribavirin depletes GTP pools and/or interferes with cap mRNA formation (Goswami et al., 1979) is not supported by several lines of evidence (Smith and Kirkpatrick, 1980; Patterson and Fernandez Larsson, 1990 ). Picornaviruses do not possess a cap structure in their mRNA, but ribavirin potently inhibits their growth. Indeed, ribavirin is active against several members of the Picornaviridae family, including poliovirus (De Clercq, 1985; Van Aerschot et al., 1989; Andersen et al., 1992) , EMC virus, FMDV (de la Torre et al., 1987) , rhinoviruses and coxsackie virus (Smith and Kirkpatrick, 1980; Andersen et al., 1992) . Ribavirin is the only antipicornavirus agent shown to cure cells from persistent picornavirus infections (de la Torre et al., 1987) . Thus, BHK cells persistently infected with FMDV were cured after passages through medium containing low concentrations of ribavirin (de la Torre et al., 1987) . Inhibition of FMDV by ribavirin in lytic infections required higher doses of the antiviral agent (de la Torre et al., 1987) . Recently, the actions of ribavirin and selenazofurin against several picornaviruses have been investigated (Andersen et al., 1992) . Prevention of poliovirus protein synthesis by ribavirin occurs when several rounds of virus replication take place (Andersen et al., 1992) , but the drug does not inhibit poliovirus protein synthesis in a single growth cycle, even if present from the beginning of infection (Almela and Carrasco, unpublished observations) . Some compounds are structurally related to ribavirin, such as selenazofurin, that also has a broad spectrum of antiviral activity, and tiazofurin (2 fl-D-ribofuranosylthiazole-4-carboxamide) that is mainly active against some RNA viruses (Srivastava et al., 1977; Huggins et al., 1984; Kirsi et al., 1984) . Bredinin, an antibiotic isolated from Eupenicillium brefeldianum, does not possess broad-spectrum antiviral activity.

These three antibiotics (Fig. 32 ) are pyrrolopyrimidine nucleosides, which are adenosine analogues. They have been isolated from different Streptomyces species: Tubercidin from Streptomyces tubercidius, toyocamycin from Streptomyces toyocaensis and sangivamycin from Streptomyces rimosus (Suhadolnik, 1967b) . All three were potent inhibitors of several rhinoviruses at drug concentrations below 1 #g/mL, but were toxic to cells at concentrations about 10-fold higher De Clercq et al., 1986) . Several tubercidin analogues have been synthesized that are much more potent against coxsackievirus De Clercq et aL, 1984) . Although these compounds may have no therapeutic utility, some of them may be very useful as inhibitors of viral RNA replication, particularly for those picornaviruses, such as cardioviruses, against which no selective inhibitors of viral RNA synthesis have been described. Thus far, we do not know how these compounds affect EMC virus, nor do we know the exact step that they block in rhinovirus or coxsackie virus growth.

Neplanocins (Fig. 33 ) are carbocyclic analogues of purine nucleosides in which the ribose moiety has been replaced by a cyclopentene ring. They have been isolated from the actinomycete Ampullaria regularis. Neplanocins, as well as other carbocyclic analogues of adenosine, have been described as potent inhibitors of S-adenosyl-L-homocysteine hydrolase, resulting in the blockade of S-adenosylmethionine-dependent methylation reactions . Neplanocins A, B and C are also broad-spectrum antiviral agents that show activity mainly against DNA and ( -)RNA viruses (De Clercq, 1985) . These agents are good inhibitors of the coxsackie virus in Vero cells. Concentrations of 40ng/mL of neplanocin A inhibited coxsackie B4 (De Clercq, 1985) , whereas higher concentrations of these agents are required to block poliovirus or coxsackie virus in HeLa cells (De Clercq, 1985) or rhinovirus in WI-38 cells (De Clercq et al., 1991) .

The carbocyclic analogues of adenosine, such as aristeromycin (C-Ado), 3-deazaadenosine (C-c3Ado) and 7-deazaadenosine (C-cTAdo) (Fig. 33) , are also active against several animal viruses, De Clercq, 1985) . C-cTAdo was more active against both viruses in HeLa cells than C-c3Ado .

Carbocyclic cytidine (C-Cyd) and cyclopentenylcytosine (Ce-Cyd) (Fig. 33 ) are broad-spectrum antiviral agents active against DNA-and RNA-containing animal viruses . Poliovirus, coxsackie virus B4 and rhinovirus 1A were not inhibited by C-Cyd, whereas they were potently blocked by Ce-Cyd (De Clercq et al., 1991) .

3-Deaza guanine (Fig. 33) , a base analogue of guanine, has good antirhinovirus activity in WI-38 cells at drug concentrations well below those that show cytotoxicity .

Several fluorinated nucleosides have been synthesized and evaluated for their antiviral potency (Van Aerschot et al., 1989) . 3'-fluoro-Y-deoxyadenosine (Fig. 32 ) was very active against poliovirus and coxsackie virus (Van Aerschot et al., 1989) . Although the action of some of these derivatives may be related to their inhibition of S-adenosyl-L-homocysteine hydrolase, the exact mode of action of 3'-fluoro-3'-deoxyadenosine remains undetermined.

Cordycepin (Y-deoxyadenosine) (Fig. 32 ) was the first natural nucleoside antibiotic isolated. It is synthesized by Cordiceps militaris and Aspergillus nidulans and inhibits the formation O f poly(A) tails in mRNAs. Cordycepin is incorporated into the 3' terminus of several RNAs and acts as a chain terminator. The antibiotic inhibits the replication of poliovirus and rhinovirus, with inhibition of the latter being particularly sensitive (Nair and Panicali, 1976) . The triphosphate inhibited incorporation of GMP by picornavirus-specific polymerase complexes in cell-free systems. RNA products synthesized in vitro lacked full-length viral RNA, indicating that RNA chain termination occurred in the presence of cordycepin (Panicali and Nair, 1978).

Amicetin (Fig. 34) , an antibiotic produced by Streptomyces fasciculatus and Streptomyces plicatus, is an inhibitor of peptide bond formation (Vhzquez, 1979) . Amicetin is active in bacteria, but is less effective against mammalian cells. It inhibits both poliovirus and EMC virus at concentrations that do not affect cultured cells (Alarc6n et al., 1984c) . Actinobolin (Fig. 34) , another translation inhibitor (V~quez, 1979) , is produced by Streptomycesfasciculatus and is also an effective antiviral agent in cell culture (Alarc6n et al., 1984c) . At concentrations of 30 or 75 #g/mL, the drug protected HeLa cells from infection by either poliovirus or EMC virus (Alarc6n and Carrasco, unpublished results 

Tetracenomycin C (Fig. 34) is an antibiotic produced by Streptomyces glaucescens. This agent blocks the growth of several animal viruses, including poliovirus and EMC virus (Alarc6n et al., 1984c) . Concentrations of 15#g/mL tetracenomycin C protected HaLa cell monolayers from infection by these two viruses (Alarc6n et al., 1984c) .

Sulfated polysaccharides and oligosaccharides inhibit viral replication at very low concentrations and show a wide spectrum of antiviral action (Alarc6n et al., 1984c; Gonz~tlez et al., 1987) . These agents have no adverse effects on cultured cells even at high drug concentrations (Alarc6n et al., 1984c; Gonzfilez et al., 1987) . Some picornaviruses, such as poliovirus, are not affected by these compounds, whereas EMC virus growth is potently blocked by carrageenan or low-MW dextran sulfate (Gonz~lez et al., 1987) . Preliminary results from our laboratory indicate that carrageenan has no effect on protein synthesis in EMC virus-infected cells, implicating a late step of EMC virus replication as the target of these agents. This result contrasts with the action of sulfated polysaccharides on herpes virus or HIV, where there is interference with an early step of infection after interaction of the virus with the host cell receptor (Gonzfilez et al., 1987; Callahan et al., 1991) .

Suramin (Fig. 35) is a polyanionic synthetic compound that inhibits several viral and cellular polyrnerases (Basu and Modak, 1985) , including the reverse transcriptase of RNA tumour viruses in cell-free systems (De Clercq, 1979; Jentsch et al., 1987) . It shows a potent antiviral effect in cultivated cells infected with different animal viruses, including EMC virus, but not poliovirus (Alarcbn et al., 1984(:; Mitsuya et al., 1984; Sola et al., 1986b; Schols et al., 1990) . Suramin had no effect on EMC virus protein synthesis even when present at early stages of infection, suggesting that it blocks a late step in the replication cycle (Alarc6n and Carrasco, unpublished results) . Trypan blue, a very nonpermeant dye, also protected cell monolayers from viral infection (Alarc6n et al., 1984c) , as occurs with other charged compounds (Balzarini et al., 1986) . In contrast with trypan blue, high concentrations of suramin had no adverse effects on either cell morphology or cell growth, even after prolonged incubation. Mice infected intravaginally with HSV-2 survived when treated with suramin (Alarc6n and Carrasco, unpublished results).

The mineral condensed ion ammonium 5-tungsto-2-antimonate (HPA 23) is a heteropolyanion that has a broad spectrum of antiviral activity and blocks EMC virus (Werner et al., 1976; Machamer et al., 1992) . A single intraperitoneal injection of HPA 23 protected mice from EMC virus (Werner et al., 1976) . Since this compound is a polyanion that also blocks reverse transcriptase (Chermann et al., 1975; Ablashi et al., 1977) , it is possible that its mode of action may be similar to that of sulfated polysaccharides. Glycyrrhizic acid (Fig. 34) is a natural compound extracted from the roots of Glycyrrhiza glabra. This compound has a broad spectrum of antiviral action (Pompei et aL, 1979; Alarc6n et al., 1984c) and blocks EMC virus, but not poliovirus, growth (Pompei et al., 1979) . Addition of this agent from the beginning of EMC virus infection allows the synthesis of viral proteins at control levels, whereas the formation of infectious and physical particles is severely diminished (Lacal and Carrasco, unpublished observations) . It is possible that this agent blocks the assembly of new EMC virions, but such an action has not yet been demonstrated.

The antibiotic tenuazonic acid (Fig. 36) was reported as an inhibitor of poliovirus growth (Miller et al., 1963) . It was later found that this compound blocks protein synthesis in mammalian cells, and it was, in fact, the first inhibitor of eukaryotic ribosomes to show selectivity in the blockade of human, as opposed to yeast ribosomes (Carrasco and V~quez, 1973). 7.5.8. N-Phenyl-N'-Arylthiourea Derivatives of N-phenyl-N'-arylthiourea (Fig. 36 ) are inhibitors of coxsackie virus, poliovirus, FMDV and rhinoviruses. Drug concentrations of approximately 2/~g/mL of N-phenyl-N'-3hydroxyphenyl-thiourea (PTU-23) reduced rhinovirus growth by one-log (Galabov et al., 1977) . The potency and antiviral index of N-phenyl-N'-arylthiourea are rather low and probably these compounds block an early step of rhinovirus replication after adsorption (Galabov et al., 1977; Galabov, 1979) . PTU-23 had activity against coxsackie infection in mice, whereas another derivative (PTU-24) was active against FMDV in mice and guinea pigs (Galabov, 1979) . 7.5.9. Dichlorobenzoate (M12325) 5-Aminosulfonyl-2,4-dichlorobenzoate (M 12325) (Fig. 36) inhibits several species of RNA-containing viruses, including echovirus and rhinovirus, but has no effect against DNA viruses (Ohnishi et al., 1982) . It has a good antiviral index in cultured cells and is effective in mice against influenza virus (Ohnishi et al., 1982). 7.5.10. G413 2-Amino-5-(2-sulfamoylphenyl)-l,3,4-thiadiazole (G413) (Fig. 36) belongs to a series of derivatives that block DNA and RNA viruses (Bonina et al., 1982) . Drug concentrations above 10 #g/mL inhibited poliovirus, echovirus and coxsackie virus. It has been suggested that G413 may bind to structural proteins and prevent virion assembly (Bonina et al., 1982) , although more profound studies on this agent are required to obtain a firm conclusion of its mode of action.

A series of water-soluble thiazolinone derivatives (IMTO) (Fig. 36 ) exhibit antiviral properties. The N-(2-dimethylamino-2-methylpropoxycarbonyl) derivative was effective against influenza and coxsackie viruses in cultured cells (Harnden et al., 1979) .

Various species of the genus Penicillium produce mycophenolic acid (Fig. 37) . This antibiotic blocks the replication of several animal viruses, including some retroviruses (Williams et al., 1968) , EMC virus and coxsackie virus Cline et al., 1969; Planterose, 1969) . It is still uncertain if this blockade is due simply to cellular toxicity. 

A new class of depsipeptides, didemnins (Fig. 37) , were isolated from a Caribbean tunicate (sea squirt, family Didemnidae) that had effects against coxsackie virus (Rinehart and Gloer, 1981) .

Although some of these compounds, particularly didemnin B, had a potent antiviral effect in cell culture, their cytotoxicity was very high and, hence, the antiviral index was poor (Rinehart and Gloer, 1981) . 7.5.14. Atropine Atropine (Fig. 37) is an antiviral substance extracted from Atropa belladonna. This agent inhibits the growth of several animal viruses (Alarcrn et al., 1984b) , including poliovirus, but not EMC virus (Alarc6n and Carrasco, unpublished results). 5.15. 910,,4 3,4-Dihydro-l-isoquinolineacetamide hydrochloride (DIQA) (Fig. 37) and some related compounds, dehydroemetine and papaverine, show some antiviral effects against picornaviruses Prince, 1968, 1970) . Only DIQA (0.1 mg/mL) was active against coxsackie virus in tissue culture (Grunberg and Prince, 1970) . Studies on the mode of action of this agent indicated that it blocked an early step of Columbia SK virus growth, different from adsorption of the virus to L cells (Murphy and Glasgow, 1970) . Probably, this early step is the entry of virions into cells (Murphy and Glasgow, 1970) . Clinical trials with DIQA have been conducted to prevent rhinovirus infection in volunteers (Togo et al., 1973a) . Oral administration did not prevent the development of colds, but the symptoms in the treated group were milder than in the placebo-treated group (Togo et aL, 1973a) .

Rifamycins A-E have been isolated from the broth of Nocardia mediterranei. Rifampicin is a chemical derivative, 3-(4-methylpiperazinoiminomethyl) rifamycin B (Fig. 37) . These antibiotics and several derivatives have activity against several animal viruses (Gurgo et al., 1982) . FMDV and poliovirus growth is inhibited by rifampicin in suspension culture, but not in cell monolayers (Grado and Ohlbaum, 1973) . Rifampicin was equally effective when added before or after virus entry. The specificity and reproducibility of this inhibition is obscure, because the same authors also found inhibition of these viruses by actinomycin D (Grado and Ohlbaum, 1973) . K-582 is a peptide antibiotic isolated from the culture filtrate of Metarhizium anisopliae, which exhibits antiviral activity against polio, influenza and Newcastle disease virus, perhaps by inducing an antiviral substance related to interferon (Sakai et al., 1983) .

Several undefined agents have been reported to block picornaviruses. Indeed, antiviral researchers are well acquainted with a numerous list of publications claiming antiviral effects with extracts, exudates, secretions, etc., from the most diverse organisms. In many instances, these crude preparations (or even purified components) owe their "antiviral activity" simply to toxicity. In some cases, however, some specific antiviral effects have been documented. Thus, helenine, perhaps a ribonucleoprotein substance, produced by Penicillium funiculosum, has effects against Columbia SK virus and SFV (Shope, 1953a, b, c) .

Extracts from different plants and fungi can interfere with the growth of poliovirus, coxsackie virus and echoviruses in cultured cells (Goulet et al., 1960) . Treatment with neurotoxoids from cobra venom interferes with poliovirus infection in monkeys and cultured cells (Sanders et al., 1953 (Sanders et al., , 1958 Miller et al., 1977) . Abalone (Haliotis refescens) is a marine animal commonly used as food in China, and its juice has some inhibitory effects on mice infected with poliovirus (Li, 1960) . Apple juice, grape juice and wines are virucidal for some viruses, including poliovirus (Konowalchuk and Speirs, 1976; Knowalchuk and Speirs, 1978) . A lipid component of milk inhibits viral growth (Sabin and Fieldsteel, 1962; Matthews et al., 1976) . The same occurs with acidic substances from pine cones and pine seed shell extracts (Fukuchi et al., 1989a; Mukoyama et al., 1991b) and with tea extracts (Green, 1949; Mukoyama et al., 1991a) . These effects perhaps relate to tannin and polyphenol content, substances that are known to be antiviral agents (Green, 1948; Kucera and Herrmann, 1967; Fukuchi et al., 1989b) .

Finally, a number of antiviral compounds have been reported to have a negligible or no effect against several picornaviruses. Such compounds include camptothecin (Horwitz et al., 1972) , gossypol (Dorsett et al., 1975) and hypericin (Tang et al., 1990) .

Picornaviruses represent one of the best understood animal virus groups in molecular terms. They were the first animal viruses containing an RNA genome that was sequenced, the first animal virus particles to be solved at atomic resolution and the first animal viruses to be totally synthesized in the test tube . The analysis of the picornavirus replication cycle, in many instances, guides work with other virus groups, although picornavirus biology also shows peculiarities typical of this virus group, e.g. the internal initiation of translation on picornavirus mRNA .

Picornavirus inhibitors have very much aided in the elucidation of the different steps of the picornavirus replication cycle at the molecular level. The growing armamentum to block given steps of picornavirus growth provides useful tools to investigate details of the molecular biology of picornaviruses. In addition, these studies provide, in some instances, new approaches to develop antiviral agents. This is the case of the recently found connection between picornavirus genome replication and membrane traffic in mammalian cells (Guinea and Carrasco, 1990; Irurzun et al., 1993) , or the finding that picornaviruses encode for viroporins (Lama and Carrasco, 1992b) , viral proteins that make pores in the membrane and modify membrane permeability , allowing the passage of nonpermeant inhibitors into virus-infected cells (Carrasco, 1978) .

The fact that some picornaviruses cause disease in humans and in animals prompted the investigation of more effective and nontoxic compounds to inhibit their replication. The elegant studies on agents that bind to virion particles and block their uncoating exemplifies not only one of the most refined analyses on the mode of action of antiviral agents, but also provides promising compounds to effectively attack the common cold in humans (Rossmann and Johnson, 1989; McKinlay et al., 1992) .

Finally, the growing interest in picornavirus inhibitors will expand the number of new antipicornavirus agents and will help to unravel the modes of action of many of them. I predict that future studies to broaden our knowledge of the exact mechanism of action of these inhibitors will provide more surprises to the molecular biology of this ever-interesting group of viruses.

Effects of 5-tungsto-2-antimoniate in oncogenic DNA and RNA virus-cell systems

0989) The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution

In: Regulation of Gene Expression in Animal Viruses

Antisense strategies: synthesis and anti-HIV activity of oligoribonucleotides and their phosphorothioate analogs

ADP-ribosylating toxins

) pppA2'p5A' blocks vesicular stomatitis virus replication in intact cells

Antiherpesvirus action of atropine

Screening for new compounds with antiherpes activity

Antirhinovirus compound 44,081 R.P. inhibits virus uncoating

Assembly and disassembly of the Golgi complex: two processes arranged in a cis-trans direction

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles

Intranasal chalcone, Ro 09-0410, as prophylaxis against rhinovirus infection in human volunteers

Suppression of colds in human volunteers challenged with rhinovirus by a new synthetic drug (R 61837)

Relationship between membrane permeability and the translation capacity of human HeLa cells studied by means of the ionophore nigericin

Permeabilization of mammalian cells to proteins by the ionophore nigericin

Molecular basis of the permeabilization of mammalian cells by ionophores

Translation of capped viral mRNAs in poliovirus-infected HeLa cells

In vitro antiviral activity of ribavirin against picornaviruses

Guanidine-resistant poliovirus mutants produce modified 37-kilodalton proteins

A functional ribonucleoprotein complex forms around the 5' end of poliovirus RNA

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region

Antivirai activity of mycophenolic acid. Studies on antiviral and antitumor antibiotics. IV

In vitro activity of R 61837, a new antirhinovirus compound

Lack of quantitative correlation between inhibition of replication of rhinoviruses by an antiviral drug and their stabilization

Eukaryotic initiation factor (elF)-4F. Implications for a role in internal initiation of translation

Polyadenylic acid sequences in the virion RNA of poliovirus and eastern equine encephalitis virus

Implications of the picornavirus capsid structure for polyprotein processing

Isolation of the structural polypeptides of foot-and-mouth disease virus and analysis of their C-terminal sequences

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14

Structural analysis of a series of antiviral agents complexed with human rhinovirus 14

Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C

Purification and properties of poliovirus double-stranded ribonucleic acid

Poliovirus-induced RNA polymerase and the effects of virus-specific inhibitors on its production

An intermediate in the synthesis of poliovirus RNA

Aurintricarboxylic acid and Evans Blue represent two different classes of anionic compounds which selectively inhibit the cytopathogenicity of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus

Purification and properties of a host cell protein required for poliovirus replication in vitro

An appraisal of the efficacy of the antiviral R 61837 in rhinovirus infections in human volunteers

Coupled translation and replication of poliovirus RNA in vitro: synthesis of functional 3D polymerase and infectious virus

Observations on the suramin-mediated inhibition of cellular and viral DNA polymerases

Activity of methisazone against RNA viruses

) 4',6-Dichloroflavan (BW683C), a new antirhinovirus compound

Effect of lysosomotropic compounds on early events in foot-and-mouth disease virus replication

Structure of the FMDV translation initiation site and of the structural proteins

Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo

Inhibition of animal virus production by means of translation inhibitors unable to penetrate normal cells

Disposition of arildone, an antiviral agent, after various routes of administration

Antiviral activity of C-5 substituted tubercidin analogues

Genetic complementation among poliovirus mutants derived from an infectious cDNA clone

Poliovirus mutant that does not selectively inhibit host cell protein synthesis

Activity of recombinant mitogillin and mitogillin immunoconjugates

Structural and functional characterization of the poliovirus replication complex

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus

Structure-activity relationships of new antiviral compounds

Proteolysis of the p220 component of the cap-binding protein complex is not sufficient for complete inhibition of host cell protein synthesis after poliovirus infection

Two modes of entry of reovirus particles into L cells

Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms--animal cells--and plant cells

Penetration of host cell membranes by adenovirus 2

Effect ofp-fluorophenylalanine on the multiplication of foot-and-mouth disease virus

Effect of Dl-ethionine on poliomyelitis virus growth in tissue culture

A novel substituted guanidine with high activity in vitro against rhinoviruses

Fatty acids stimulate phosphatidyicholine synthesis and CTP:choline-phosphate cytidylyltransferase in type II pneumocytes isolated from adult rat lung

Kinetics of synthesis and cleavage of encephalomyocarditis virus-specific proteins

Effect of ariidone on modifications of poliovirus in vitro

Action of guanidine on the replication of poliovirus RNA

Distribution and translation of poliovirus RNA in guanidine-treated cells

Membranous structures associated with translation and transcription of poliovirus RNA

Characterization of poliovirus-specific structures associated with cytoplasmic membranes

The role of cytoplasmic membranes in poliovirus biosynthesis

Guanidine and 2-(a-hydroxybenzyl)-benzimidazole (HBB): selective inhibitors of picornavirus multiplication

Dextran sulfate blocks antibody binding to the principal neutralizing domain of human immunodeficiency virus type l without interfering with gpl20-CD4 interactions

The inhibition of cell functions after viral infection. A proposed general mechanism

Membrane leakiness after viral infection and a new approach to the development of antiviral agents

Modification of membrane permeability induced by animal viruses early in infection

Mechanisms of virus-induced cell toxicity: a general overview

The regulation of translation in picornavirus infected cells

Modification of membrane permeability in vaccinia virus-infected cells

New approaches to antiviral agents

Regulation of translation of eukaryotic virus mRNAs

Modification of membrane permeability by animal viruses

Modification of membrane permeability by animal viruses

Sodium ions and the shut-off of host cell protein synthesis by picornaviruses

Molecular biology of animal virus infection

Differences in eukaryotic ribosomes detected by the selective action of an antibiotic

Viral translation inhibitors

Molecular bases for the action and selectivity of nucleoside antibiotics

Effect of lysosomotropic agents on the foot-and-mouth disease virus replication

Early steps in FMDV replication: further analysis on the effects of chloroquine

Action of 3-methylquercetin on poliovirus RNA replication

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus

3-Methylquercetin is a potent and selective inhibitor of poliovirus RNA synthesis

Eukaryotic start and stop translation sites

Human rhinovirus 14 complexed with antiviral compound R 61837

Phosphorothioate analogues of (2'-5')(A)4: agonist and antagonist activities in intact cells

Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection

Assembly and polarized release of punta toro virus and effects of brefeldin A

0975) Inhibition of RNA-dependent DNA polymerase of murine oncornaviruses by ammonium-5-tungsto-2-antimoniate

Brefeldin A arrests the maduration and egress of herpes simplex virus during infection

RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3D p°l

Myristilation of picornavirus capsid protein VP4 and its structural significance

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus

Potential secondary and tertiary structure in the genomic RNA of foot-and-mouth disease virus

In vitro antiviral activity of mycophenolic acid and its reversal by guanine-type compounds

A full-length murine 2-5A synthetase eDNA transfected in NIH-3T3 ceils impairs EMCV but not VSV replication

Isolation of a monoclonal antibody that blocks attachment of the major group of human rhinoviruses

Activities and mechanisms of action of halogen-substituted flavonoids against poliovirus type 2 infection in vitro

Selective inhibition of protein synthesis in virus-infected mammalian cells

High-efficiency receptor-mediated delivery of small and large (48 kilobase) gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles

Incorporation of C18 polyunsaturated fatty acids into L cells phospholipids under normal conditions and during infection with Venezuelan equine encephalitis virus (VEE-arbovirus)

Early events in cell-animal virus interactions

Electron microscopic study of the formation of poliovirus

Purification of host factor required for in vitro transcription of poliovirus RNA

Dependence of the activity of the poliovirus replicase on the host cell protein

Guanidine derivatives in medicine

The effect of poliovirus proteinase 2Apro expression on cellular metabolism. Inhibition of DNA replication, RNA polymerase II transcription, and translation

Absence of significant antiviral effects of beta-gammamethylene GTP on encephalomyocarditis virus infection of L cells and mice

Suramin: a potent inhibitor of the reverse transcriptase of RNA tumor viruses

Antiviral and antimetabolic activities of neplanocins

Broad-spectrum antiviral activity of adenosine analogues

Antirhinovirus activity of purine nucleoside analogs

Broad-spectrum antiviral activity of carbodine, the carbocyclic analogue of cytidine

3-Dihydroxypropyl)adenine: an aliphatic nucleoside analog with broad-spectrum antiviral activity

Broad-spectrum antiviral activity of the carbocyclic analog of 3-deazaadenosine

Broad-spectrum antiviral and cytocidal activity of cyclopentenylcytosine, a carbocyclic nucleoside targeted at CTP syntbetase

Antiviral activity and possible mechanisms of action of oligonucleotides-poly(l-lysine) conjugates targeted to vesicular stomatitis virus mRNA and genomic RNA

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro

Very high frequency of reversion to guanidine resistance in clonal pools of guanidine-dependent type 1 poliovirus

Inhibition of rhinovirus replication in organ culture by a potential antiviral drug

The Biology of the Interferon System

Synthesis and anti-rhinovirus IB activity of oxazolinylflavans

Correlation of pocket size and shape with antiviral activity

Isoxazoles with antipicornavirus activity

Dihydro-2-oxazolyl)phenoxy]alkyl]isoxazoles. Inhibitors of picornavirus uncoating

Antipicornavirus compounds: use of rational drug design and molecular modelling

Inhibitors of picornavirus uncoating as antiviral agents

Antiviral activity of some b-diketones. 2. Aryloxy alkyl diketones. In vitro activity against both RNA and DNA viruses

Antiviral activity of some b-diketones. 1. Aryl alkyl diketones. In vitro activity against both RNA and DNA viruses

A model for compounds active against human rhinovirus-14 based on X-ray crystallography data

Review article initial stages in infection with animal viruses

Suppression of the inhibitory action of guanidine on virus multiplication by some amino acids

RNA virus evolution and the control of viral disease

Genetic variation and quasi-species

Brefeldin A redistributes resident and itinerant Golgi proteins to the endoplasmic reticulum

Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein

Letter: Antiviral activity of gossypol and apogossypol

Rates of spontaneous mutation among RNA viruses

Inhibitory effect of modified bafilomycins and concanamycins on P-and V-type adenosinetriphosphatases

Entry and release of poliovirus as observed by electron microscopy of cultured cells

Staphylococcal delta toxin stimulates endogenous phospholipase A2 activity and prostaglandin synthesis in fibroblasts

Alteration of the intracellular energetic and ionic conditions by mengovirus infection of Ehrlich ascites tumor cells and its influence on protein synthesis in the midphase of infection

Successful treatment of entrovirus-infected mice by 2-(a-hydroxybenzil)-benzimidazole and guanidine

Selective inhibition of uncoating of echovirus 12 by rhodanine. A study on early virus-cell interactions

Benzimidazoles

Antiviral agents against picornaviruses

Cytoplasmic localization of the uncoating of picornaviruses

Isolation and characterization of an arildone-resistant poliovirus 2 mutant with an altered capsid protein VPI

On the mechanism of selective inhibition of enterovirus multiplication by 2-(alpha-hydroxybenzyl)-benzimidazole

Inhibition of Enterovirus ribonucleic acid synthesis by 2-(alpha-hydroxybenzyl)-benzimidazole

Drug dependence of entroviruses: variants of coxsackie A9 and ECHO 13 viruses that require 2-(a-hydroxybenzyl)-benzimidazole for growth

Coxsackie A9 virus: mutation from drug dependence to drug independence

The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins

RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes

Analogs of (A2'p)nA. Correlation of structure of analogs of ppp(A2'p)2A and (A2'p)2A with stability and biological activity

Stereoconfiguration markedly affects the biochemical and biological properties of phosphorothioate analogs of 2-5A core, (A2'p5')2A

Comparison of replication complexes synthesizing poliovirus RNA

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex

Eclipse products of poliovirus after cold-synchronized infection of HeLa cells

Foot-andmouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3

Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state

Permeabilized cells

Early interactions between poliovirus and ERK cells. Some observations on the nature and significance of the rejected particles

Ribavirin: a clinical overview

Ribavirin is an inhibitor of human immunodeficiency virus reverse transcriptase

Viral infection permeabilizes mammalian cells to protein toxins

Structural factors that control conformational transitions and serotype specificity in type 3 poliovirus

Adenovirus-induced release of epidermal growth factor and pseudomonas toxin into the cytosol of KB cells during receptor-mediated endocytosis

Poliovirus-specific primer-dependent RNA polymerase able to copy poly(A)

Covalent linkage of a protein to a defined nucleotide sequence at the Y-terminus of virion and replicative intermediate RNAs of poliovirus

Isolation of a soluble and template-dependent poliovirus RNA polymerase that copies virion RNA in vitro

A common mode of antiviral action for ammonium ions and various amines

Contact inactivation of RNA and DNA viruses by N-methyl isatin beta-thiosemicarbazone and CuSO4

Prevention of rhinovirus and poliovirus uncoating by WIN 51711, a new antiviral drug

Cell-induced conformational change in poliovirus: externalization of the amino terminus of VP1 is responsible for liposome binding

Basis for variable response of arboviruses to guanidine treatment

Brefeldin A causes disassembly of the Goigi complex and accumulation of secretory proteins in the endoplasmic reticulum

Inhibition of herpes virus infection by pine cone antitumor substances

Inhibition of herpes simplex virus infection by tannins and related compounds

Thiourea derivatives as specific inhibitors of picorna viruses

Antiviral activity of N-phenyi-N'-arylthiourea derivatives against some rhinoviruses

Alteration of intracellular monovalent cation concentrations by a poliovirus mutant which encodes a defective 2A protease

Na + and K ÷ concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells

C-nucleosides: aspects of chemistry and mode of action

Selective inhibition of poliovirus growth by D-penicillamine in vitro

Acid-induced structural changes in human rhinovirus 14: possible role in uncoating

In vitro synthesis of poliovirus ribonucleic acid: role of the replicative intermediate

The poliovirus replication complex: site for synthesis of poliovirus RNA

Polysaccharides as antiviral agents: antiviral activity of carrageenan

3,4-Dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile inhibits poliovirus uncoating

Animal viruses promote the entry of polysaccharides with antiviral activity into cells

Flavonoids: potent inhibitors of poliovirus RNA synthesis

Inhibition of human immunodeficiency virus replication by antisense oligodeoxynucleotides

A new superfamily of putative NTP-binding domains encoded by genomes of small DNA and RNA viruses

The broad spectrum antiviral agent ribavirin inhibits capping of mRNA

Mechanism of inhibition of herpesvirus growth by 2'-5'-linked trimer of 9-b-d-xylofuranosyladenine

Differential and specific inhibition of ECHO viruses by plant extracts

The effect of rifampicin, actinomycin D and mitomycin C on poliovirus and foot-and-mouth disease virus replication

Three-dimensional structure of Theiler virus

Peptidyl diazomethyl ketones are specific inactivators of thiol

Inhibition of influenza virus by tannic acid

Inhibition of influenza virus by extracts of tea

The major human rhinovirus receptor is ICAM-I

Histone H3 modification in BHK cells infected with foot-and-mouth disease virus

Kinetics of poliovirus uncoating in HeLa cells in a nonacidic environment

Stabilization of human rhinovirus serotype 2 against pH-induced conformational change by antiviral compounds

The antiviral activity of 3,4-dihydro-l-isoquinolineacetamide hydrochloride in vitro, in ovo, and in small laboratory animals (33335)

Experimental methodology and the search for effective antiviral agents

Phospholipid biosynthesis and poliovirus genome replication, two coupled phenomena

Effects of fatty acids on lipid synthesis and viral RNA replication in poliovirus-infected cells

Modification of phospholipase C and phospholipase A2 activities during poliovirus infection

A plasma membrane component able to bind and alter virions of poliovirus type 1: studies on cell-free alteration using a simplified assay

Virazole (Ribavirin)

Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A

Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

Aspects of the molecular biology of poliovirus replication

The Flavonoids: Advances in Research Since

Easily hydrolyzable, water-soluble derivatives of( + / -)-alpha-5-[1-(indoi-3-yl)ethyl]-2-methylamino-delta-2-thiazoline-4-one, a novel antiviral compound

Single-chain ribosome inactivating proteins from plants depurinate Escherichia coli 23S ribosomal RNA

Analogues and analogue inhibitors of ppp(A2'p)nA. Their stability and biological activity

Clinical applications of antiviral agents for chemoprophylaxis and therapy of respiratory viral infections

Jr (1982) Prophylactic activity of intranasal enviroxime against experimentally induced rhinovirus type 39 infection

Safety and efficacy of intranasal pirodavir (R 77975) in experimental rhinovirus infection

Modification of experimental rhinovirus infection by receptor blockade

Proteolytic processing of polyproteins in the replication of RNA viruses

Viral proteases as targets for chemotherapeutic intervention

Maturation of poliovirus capsid proteins

Inhibition by brefeldin A of a (3olgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF

Separation and quantitation of intracellular forms of poliovirus RNA by agarose gel electrophoresis

Primaquine blocks transport by inhibiting the formation of functional transport vesicles. Studies in a cell-free assay of protein transport through the (3olgi apparatus

Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes

Three-dimensional structure of poliovirus at 2.9A resolution

RNA virus populations as quasispecies

Evidence for poliovirus-induced cytoplasmic alkalinization in HeLa cells

Formation of rhinovirus-soluble ICAM-I complexes and eonformational changes in the virion

Antiviral action of camptothecin

Synergistic antiviral effects of ribavirin and the C-nucleoside analogs tiazofurin and selenazofurin against togaviruses, bunyaviruses, and arenaviruses

Brefeldin A and the endocytic pathway: possible implications for membrane traffic and sorting

Complete nucleotide sequence of the genome of coxsackievirus B1

Inhibition of cleavage of Moloney leukemia virus gag and env coded precursor polyproteins by cerulenin

Chemical modification potentiates the biological activities of 2-5A and its congeners

Involvement of membrane traffic in the replication of poliovirus genomes: effects of Brefeldin A

Enhancement of phospholipase C activity during poliovirus infection

Pokeweed antiviral protein: ribosome inactivation and therapeutic applications

Direct and specific inactivation of rhinovirus by chalcone Ro 09-0410

Molecular basis of drug resistance to new antirhinovirus agents

Antipicornavirus flavone Ro 09-0179

The novel mechanism of initiation of picornavirus RNA translation

Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2a does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells

Further evidence on the formation of poliovirus proteins

Polypeptide cleavages in the formation of poliovirus proteins

Biochemical and genetic evidence for a pseudoknot structure at the 3t terminus of the poliovirus RNA genome and its role in viral RNA amplification

Towards gene-inhibition therapy: a review of progress and prospects in the field of antiviral antisense nucleic acids and ribozymes

A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation

Inhibition of human immunodeficiency virus type I reverse transcriptase by suramin-related compounds

Plant and fungal protein and glycoprotein toxins inhibiting eukaryote protein synthesis

Poliovirus infection results in structural alteration of a microtubule-associated protein

Three poliovirus 2B mutants exhibit noncomplementable defects in viral RNA amplification and display dosage-dependent dominance over wild-type poliovirus

Clearance of a persistent human enterovirus infection of the mouse central nervous system by the antiviral agent disoxaril

Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis

Ion channels in icosahedral virus: a comparative analysis of the structures and binding sites at their fivefold axes

Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism

Neutralization of poliovirus by cell receptors expressed in insect cells

Poliovirus mutants resistant to neutralization with soluble cell receptors

Synthesis of vaccinia viral antigens in HeLa cells in the presence of isatin beta-thiosemicarbazone

Antirhinovirus activity of 6-anilino-9-benzyl-2-chloro-9H-purines

In vitro antiviral activity of the 6-substituted 2-(Y,4'-dichlorophenoxy)-2H-pyrano

In vitro and in vivo antipicornavirus activity of some phenoxypyridinecarbonitriles

Mechanism of action of the antiviral compound MDL 20,610

The antiviral action of interferon

Virus protein synthesis in animal cell-free systems: nature of the products synthesized in response to ribonucleic acid of encephalomyocarditis virus

The antiviral effects of the interferons and their inhibition

Method for preparing specific inhibitors of virus-specific proteases

A comparison of the anti-rhinoviral drug binding pocket in HRV14 and HRV1A

Antiviral activity of arildone on deoxyribonucleic acid and ribonucleic acid viruses

Crystal structure of human rhinovirus serotype IA (HRV1A)

Genetic analysis of picornavirus. Current Opinion Genetics Develop

Broad-spectrum synergistic antiviral activity of selenazofurin and ribavirin

Primary structure, gene organization and polypeptide expression of poliovirus RNA

Brefeldin A: insights into the control of membrane traffic and organelle structure

Antiviral effects of a thioi protease inhibitor on foot-and-mouth disease virus

Antiviral effect of apple beverages

The Molecular Biology of Poliovirus

Unsaturated free fatty acids inactivate animal enveloped viruses

The poliovirus receptor protein is produced both as membrane-bound and secreted forms

Functional domains of the poliovirus receptor

A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain

Transgenic mice susceptible to poliovirus

Beneficial effect of new thiol protease inhibitors, epoxide derivatives, on dystrophic mice

Virus inactivation by grapes and wines

Virus-specified protease in poliovirus-infected HeLa cells

Cleavage of viral precursor proteins in vivo and in vitro

Viral protein cleavages: target for viral therapy

Strategies to inhibit viral polyprotein cleavages

Viral protein cleavages as a drug design target

Cystatin, a protein inhibitor of cysteine proteases alters viral protein cleavages in infected human cells

Zinc ions inhibit replication of rhinoviruses

Viral therapy: prospects for protease inhibitors

The scanning model for translation: an update

Regulation of translation in eukaryotic systems

Structural refinement and analysis of Mengo virus

Chloroquine induces empty capsid formation during poliovirus eclipse

Compartmentalization of subviral particles during poliovirus eclipse in HeLa cells

Antiviral substances in plants of the mint family (Labiatae). I. Tannin of Melissa oJficinalis. II. Non-tannin polyphenol of Melissa officinalis

Preliminary studies of the mode of action of arildone, a novel antiviral agent

Studies on the role of the 2'-5'-oligoadenylate syntbetase-RNase L pathway in beta interferon-mediated inhibition of encephalomyocarditis virus replication

Effects of ouabain on the anticellular and antiviral activities of human and mouse interferon

Relationship between membrane integrity and the inhibition of host translation in virus-infected mammalian cells. Comparative studies between encephalomyocarditis virus and poliovirus

Modification of membrane permeability in poliovirus-infected HeLa cells: effect of guanidine

Antiviral effects of hygromycin B, a translation inhibitor nonpermeant to uninfected cells

Antibiotics that specifically block translation in virus-infected cells

Specific inhibition of poliovirus induced blockade of cell protein synthesis by a thiopyrimidine derivative

Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins

Inducible expression of a toxic poliovirus membrane protein in Escherichia coil Comparative studies using different expression systems based on T7 promoters

Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A

Cloning and inducible synthesis of poliovirus nonstructural proteins

Interactions of the elF-4F subunits in the yeast Saccharomyces cerevisiae

Immunological evidence for the in vivo occurrence of (2'-5')adenylyladenosine oligonucleotides in eukaryotes and prokaryotes

Picornavirus protein processing---enzymes, substrates, and genetic regulation

The interferon-induced double-stranded RNA-activated human p68 protein kinase inhibits the replication of vaccinia virus

Poliovirus-mediated entry of pokeweed antiviral protein

A protein covalently linked to poliovirus genome RNA

Inhibition of influenza virus replication by phosphorothioate oligodeoxynucleotides

A fraction of the mRNA 5' cap-binding protein, eukaryotic initiation factor 4E, localizes to the nucleus

The recognition event between virus and host cell receptor: a target for antiviral agents

Antibody-targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication

Topical enviroxime against rhinovirus infection

Inhibition of viruses, tumors, and pathogenic microorganisms by isatin beta-thiosemicarbazone and other thiosemicarbazones

Effect of p-fluorophenylalanine and puromycin of the replication of poliovirus

Induction of an antiviral state by interferon in the absence of elevated levels of 2,5-oligo(A) syntbetase and eIF-2 kinase

Antimicrobial effect of abalone juice

Isolation of poliovirus 2C mutants defective in viral RNA synthesis

An intragenic revertant of a poliovirus 2C mutant has an uncoating defect

Bidirectional membrane traffic between the endoplasmic reticulum and Golgi apparatus

Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from Golgi to ER

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic

Persistent infection of human erythroblastoid cells by poliovirus

Relationship of p220 cleavage during picornavirus infection to 2A proteinase sequencing

Antagonism of the guanidine interference with poliovirus replication by simple methylated and ethylated compounds

Guanidine conditional infectivity of ribonucleic acid extracted from a strain of guanidine-dependent polio-I virus

Early alteration of poliovirus in infected cells and its specific inhibition

Cell type determines the relative proportions of ( -) and ( + ) strand RNA during poliovirus replication

Lack of evidence for strand-specific inhibition of poliovirus RNA synthesis by 3-methylquercetin

Cation content in poliovirus-infected HeLa cells

The atomic structure of Mengo virus at 3.0 A resolution

The specific effectors of viral development

The E1 glycoprotein of an avian coronavirus is targeted to the cis Golgi complex

Different pH requirements for entry of the two picornaviruses, human rhinovirus 2 and murine encephalomyocarditis virus

Requirements for entry of poliovirus RNA into cells at low pH

Mechanism of entry into the cytosol of poliovirus type 1: requirement for low pH

A soluble form of intercellular adhesion molecule-1 inhibits rhinovirus infection

Virus entry into animal cells

Phosphorodithioate DNA as a potential therapeutic drug

Efficient neutralization and disruption of rhinovirus by chimeric ICAM-1/immunoglobulin molecules

Antibody-complexed foot-and-mouth disease virus, but not poliovirus, can infect normally insusceptible cells via the Fc receptor

The antiviral activity of a triazinoindole (SK&F 30097) (36163)

Antiviral activity in milk of possible clinical importance

Inhibition of poliovirus RNA synthesis by brefeldin A

Dextrin sulphate and fucoidan are potent inhibitors of HIV infection in vitro

Antiviral action of gliotoxin

WIN 51711, a new systematically active broad-spectrum antipicornavirus agent

Treatment of the picornavirus common cold by inhibitors of viral uncoating and attachment

Rational design of antiviral agents

Oral efficacy of WIN 51711 in mice infected with human poliovirus

Viral inhibition of the interferon system

Inhibition of uncoating of poliovirus by arildone, a new antiviral drug

In vitro mutational analysis of cis-acting RNA translational elements within the poliovirus type 2 5' untranslated region

Studies on the mechanism of internal initiation of translation on poliovirus RNA

La autoantigen enhances and corrects aberrant translation of poliovirus RNA in reticulocyte lysate

Cell-penetrating inhibitors of calpain

Cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily

Effect of o-penicillamine on poliovirus replication in HeLa cells

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth

Conformational alteration of Sandbis virion glycoproteins induced by heat, reducing agents, or low pH

Epipolythiopiperazinedione antibiotics from Penicillium turbatum

Antiviral activity of tenuazonic acid

Inhibition of virus-induced plaque formation by a toxic derivative of purified cobra neurotoxin

Specific inhibition of viral ribonucleic acid replication by gliotoxin

The molecular biology of poliovaccines

Molecular biology and the control of viral vaccines

Monoclonal antibodies which block cellular receptors of poliovirus

Novel blockade by brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes

Suramin protection of T cells in vitro against infectivity and cytopathic effect of HTLV-III

Molecular mechanisms of action of ribavirin

Capsid protein VP4 of poliovirus is N-myristoylated

Inhibition of poliovirus replication by N-methylisatin-beta-4' :4'-dibutylthiosemicarbazone

Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA

Melittin stimulates incorporation and degradation of sphyngomyelin in synaptosomal plasma membranes

Isatin-beta-thiosemicarbazone causes premature cessation of vaccinia virus-induced late post-replicative polypeptide synthesis

Cerulenin--an inhibitor of lipid synthesis-blocks vesicular stomatitis virus RNA replication

Lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection

Entry of poliovirus into cells does not require a iow-pH step

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis

Conformational change in the floor of the human rhinovirus canyon blocks adsorption to HeLa cell receptors

The thiosemicarbazones

The reversion of guanidine inhibition of poliovirus synthesis

Failure of oral 4',6-dichloroflavan to protect against rhinovirus infection in man

Therapeutic activity of enviroxime against rhinovirus infection in volunteers

Guanidine-selected mutants of poliovirus: mapping of point mutations to polypeptide 2C

Guanidine-dependent mutants of poliovirus: identification of three classes with different growth requirements

Production of guanidine-resistant and -dependent poliovirus mutants from cloned cDNA: mutations in polypeptide 2C are directly responsible for altered guanidine sensitivity

The antiviral effect of a triazinoindole (SK&F 40491) in rhinovirus infected gibbons

Influenza virus M2 protein has ion channel activity

Antiviral and cytotoxic effects of mycophenolic acid

Pseudoknots: a new motif in the RNA game

Stabilization of poliovirus by cystine

Glycyrrhizic acid inhibits virus growth and inactivates virus particles

Activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) against picornaviruses in vitro

Picornaviral structure and assembly

Cell receptors for picornaviruses

Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome

Cloned poliovirus complementary DNA is infectious in mammalian cells

Evolutionary families of peptidases

The assessment of antirhinovirus compounds with clinical potential

Transgenic mice expressing a human poliovirus receptor: a new model for poliomyelitis

Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice

Cap recognition and the entry of mRNA into the protein synthesis initiation cycle

Regulation of eukaryotic protein synthesis by initiation factors

Double-stranded RNA-dependent protein kinase and 2-5A system are both activated in interferon-treated, encephalomyocarditis virus-infected HeLa cells

Poliovirus RNA replication

Antiviral effect of guanidine

Didemnins: antiviral and antitumor depsipeptides from a Caribbean tunicate

The importance of IMP dehydrogenase inhibition in the broad spectrum antiviral activity of ribavirin and sclenazofurin

Gliotoxin: Inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3D p°~

Poliovirus protein 2C has ATPase and GTPase activities

A comparison of WIN 51711 and R 78206 as stabilizers of poliovirus virions and procapsids

Echovirus 12-induced host cell shutoff is prevented by rhodanine

Early processes of echovirus 12-infection: elution, penetration, and uncoating under the influence of rhodanine

Antiviral agents targeted to interact with viral capsid proteins and a possible application to human immunodeficiency virus

The structure of antiviral agents that inhibit uncoating when complexed with viral capsids

The canyon hypothesis. Hiding the host cell receptor attachment site on a viral surface from immune surveillance

Icosahedral RNA virus structure

Structure of a human common cold virus and functional relationship to other picornaviruses

Molecular dissection of the secretory pathway

Systematic nomenclature of picornavirus proteins

Antipoliomyelitic activity of human and bovine colostrum and milk

Selective and reversible inhibition of initiation of protein synthesis in mammalian cells

Interferon induction in mice by oral administration of K-582, a new peptide antibiotic

Neurotoxoid interference with two human strains of poliomyelitis in Rhesus monkeys

Neurotoxoid interference in Macacus rhesus infected intramuscularly with poliovirus

Role of Y-end sequences in infectivity of poliovirus transcripts made in vitro

A poliovirus temperature-sensitive RNA synthesis mutant located in a noncoding region of the genome

Poliovirus genetics

Guanidine-resistant mutants of aphthovirus induce the synthesis of an altered nonstructural polypeptide, P34

Cordycepin analogues of 2-5A and its derivatives. Chemical synthesis and biological activity

-Hydroxyethoxymethyl) guanine activity against viruses of the herpes group

Quantitative study of the formation of poliovirus antigens in infected HeLa cells

Prophylactic efficacy of WIN 54954 in prevention of experimental human coxsackievirus A21 infection and illness

Cerulenin blocks fatty acid acylation of glycoproteins and inhibits vesicular stomatitis and Sindbis virus particle formation

The proton pump ATPase of lysosomes and related organelles of the vacuolar apparatus

Dextran sulfate and other polyanionic anti-HIV compounds specifically interact with the viral gp 120 glycoprotein expressed by T-cells persistently infected with HIV-I

Successful competition in translation by the flavivirus Kunjin with poliovirus during co-infections in Vero cells

Inhibitors of lysosomal function

Interferon and its inducers

A chimeric plasmid from eDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive

Replication of the poliovirus genome

Mechanisms of action and pharmacology: chemical agents

WIN 52035-2 inhibits both attachment and eclipse of human rhinovirus 14

An antiviral substance from Penicillium funiculosum: III. General properties and characteristics of helenine

An antiviral substance from Penicillium funiculosum: I. Effect upon infection in mice with Swine influenza virus and Columbia SK encephalomyelitis virus

An antiviral substance from Penicilliumfuniculosum: II. Effect of helenine upon infection in mice with Semliki Forest virus

Progress with antiviral agents

Ribavirin: an antiviral agent

Failure of guanidine and 2-(alpha-hydroxybenzyl)bcnzimidazole to inhibit replication of hepatitis A virus in vitro

Control of the ppp(A2'p)nA system in HeLa cells. Effects of interferon and virus infection

Experimental transovarial transmission of Kyasanur Forest disease virus in Haemaphysalis spinigera

Structure and stereochemistry of thysanone: a novel human rhinovirus 3C-protease inhibitor from Thysanophora penicilloides

Spiro indolinone beta-lactams, inhibitors of poliovirus and rhinovirus 3c-proteinases

Ribavirin: A Broad Spectrum Antiviral Agent

The site of attachment in human rhinovirus 14 for antiviral agents that inhibit uncoating

Preliminary crystallographic analysis of coxsackievirus A9

Inactivation and inhibition of African swine fever virus by monoolein-monolinolein and gamma-linolenyl alcohol

New agents active against African swine fever virus

Poliovirus translation

Picornavirus RNA translation continues to surprise

Polyadenylic acid on poliovirus RNA. II. Poly(A) on intracellular RNAs

Polyadenylic acid on poliovirus RNA IV. Poly(U) in replicative intermediate and double-stranded RNA

Studies on the function of polyadenylic acid on poliovirus RNA

Synthesis and antiviral activity of certain thiazole C-nucleosides

Interferon-induced proteins and the antiviral state

Structure, function and evolution of picornaviruses

The arrangement of the immunoglobulin-like domains of ICAM-I and the binding sites for LFA-1 and rhinovirus

A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses

Antiviral effects of single-stranded polynucleotides and their mode of action

Investigation of the anti-viral mechanism of poly I and poly C against encephalomyocarditis virus infection in the absence of interferon induction in mice

In vivo antiviral activity of polynucleotide mimics of strategic regions in viral RNA

Inactivation and heat stabilization of poliovirus by 2-thiouracil

Concentrations in serum and urinary excretion of guanidine, 1-methylguanidine and 1,1-dimethylguanidine in renal failure

Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide

A second protease of foot-and-mouth disease virus

Mechanism of action of l-/~-D-ribofuranosyl-l,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent

New antiviral compounds

Induction of interferon with low molecular weight compounds: fluorenone esters, ethers (Tilorone), and pyramidinones

Intracellular digestion of reovirus particles requires a low pH and is an essential step in the viral infectious cycle

Evidence for large precursor proteins in poliovirus synthesis

Determination of the gene sequence of poliovirus with pactamycin

Inhibition of cleavage of large poliovirus-specific precursor proteins in infected HeLa cells by inhibitors of proteolytic enzymes

Role of vesicles during adenovirus 2 internalization into HeLa cells

Effect of pactamycin on synthesis of poliovirus proteins: a method for genetic mapping

Initiation of poliovirus plus-strand RNA synthesis in a membrane complex of infected HeLa cells

Membrane-dependent uridylylation of the genome-linked protein VPg of poliovirus

Halobenzimidazole ribosides and RNA synthesis of cells and viruses

Antiviral activity of brefeidin A and verrucarin A

Virucidal activity of hypericin against enveloped and non-enveloped DNA and RNA viruses

Recent progress in interferon research: molecular mechanisms of regulation, action, and virus circumvention

Guanidine inhibition of poliovirus growth. Partial elimination by protease antagonists and low temperature

Inhibition of poliovirus polymerase by guanidine in vitro

Effect of guanidine on the growth of LSc poliovirus

Guanidine

Cowpea mosaic virus middle component RNA contains a sequence that allows internal binding of ribosomes and that requires eukaryotic initiation factor 4F for optimal translation

Inhibition of an early stage of rhinovirus replication by dichloroflavan (BW683C)

Self-catalyzed linkage of poliovirus terminal protein VPg to poliovirus RNA

Antiviral effect of 3,4-dihydro-l-isoquinolineacetamide hydrochloride in experimental human rhinovirus infection

Failure of a 3-substituted triazinoindole in the prevention of experimental human rhinovirus infection

cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1

Antiviral action of dipyridamole and its derivatives against influenza virus A

Antiviral activity and mechanism of action of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860)

A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein

Antiviral activity of N,N'-dimethyl-epidithiapiprazinedione, a synthetic compound related to the gliotoxins, LL-S88alpha and beta, chetomin and the sporidesmins

Mechanism of action of gliotoxin: elimination of activity by sulfhydryl compounds

Intracellular localization of poliovirus RNA by in situ hybridization at the ultrastructural level using single-stranded riboprobes

Efficacy of oral WIN 54954 for prophylaxis of experimental rhinovirus infection

Antirhinovirus drugs. In: Targets for the Design of Antiviral Agents

Effects of brefeldin A on the processing of viral envelope glycoproteins in murine erythroleukemia cells

Cell killing by enveloped RNA viruses

Post-translational modifications of poliovirus proteins

Degradation of cellular proteins during poliovirus infection: studies by two-dimensional gel electrophoresis

Inhibition of poliovirus replication by a plant antiviral peptide

Synthesis and antiviral activity evaluation of 3'-fluoro-Y-deoxyribonucleosides: broad-spectrum antiviral activity of 3'-fluoro-Ydeoxyandenosine

Plant antiviral agents; V. 3-Methoxyflavones as potent inhibitors of viral-induced block of cell synthesis

Infectious entry pathway of adenovirus type 2

Inhibitors of Protein Biosynthesis

Effect of treatment with certain flavonoids on mengo virus-induced encephalitis in mice

2-5A Synthetase activity induced by interferon a, b and g in human cell lines differing in their sensitivity to the anticellular and antiviral activities of these interferons

Antiviral activity of 3-methoxyftavones

Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells

Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle

Coupling of adenovirus to transferrin-polylysine-DNA complexes greatly enhances receptor-mediated gene delivery and expression of transfected genes

Effect of ammonium 5-tungsto-2-antimoniate on encephalomyocarditis and vesicular stomatitis virus infections in mice

Effect of brefeldin A on alphaherpesvirus membrane protein glycosylation and virus egress

Viral and cellular membrane fusion proteins

Specificity in initiation of protein synthesis in a fractionated mammalian cell-free system

The structure and function of the hemagglutinin membrane glycoprotein of influenza virus

Natural occurrence of 2-5A in interferon-treated EMC virus-infected L cells

Synthesis and breakdown of pppA2'p5'p5'A and transient inhibition of protein synthesis in extracts from interferon-treated and control cells

Micophenolic acid: antiviral and antitumor properties

Molecular events leading to picornavirus genome replication

Design, synthesis, and broad spectrum antiviral activity of 1-fl-o-ribofuranosyl-l,2,4-triazole-3-carboxamide and related nucleosides

An electron microscope study of the proteins attached to polio virus RNA and its replicative form (RF)

Amplifiable messenger RNA

Eukaryotic initiation factor 3 is required for poliovirus 2A protease-induced cleavage of the p220 component of eukaryotic initiation factor 4F

Relationship of eukaryotic initiation factor 3 to poliovirus-induced p220 cleavage activity

Activity against rhinoviruses, toxicity, and delivery in aerosol of enviroxime in liposomes

Action of the active metabolites of tiazofurin and ribavirin on purified IMP dehydrogenase

Macromolecules can penetrate the host cell membrane during the early period of incubation with HVJ (Sendai virus)

Inhibition of poliovirus by effect of a methylthiopyrimidine derivative

Three-dimensional structure of a mouse-adapted type 2/type 1 poliovirus chimera

Polyadenylic acid at the 3'-terminus of poliovirus RNA

Transferrinfection: a highly efficient way to express gene constructs in eukaryotic cells

Inhibition of poliovirus uncoating by disoxaril (WIN 51711)

Entry of poliovirus type 1 and Mouse Elberfeld (ME) virus into HEp-2 cells: receptor-mediated endocytosis and endosomal or lysosomal uncoating

Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells

Studies on 44 081 R.P., a new antirhinovirus compound, in cell cultures and in volunteers

Intravesicular acidification correlates with binding of ADP-ribosylation factor to microsomal membranes

Structure determination of antiviral compound SCH 38057 complexed with human rhinovirus 14

Expression cloning of 2-5A-dependent RNAase: a uniquely regulated mediator of interferon action

Screening of antiviral drugs for hepadna virus infection in Pekin ducks: a review

Acknowledgements--I wish to thank those who sent me their reprints and preprints, and I look forward to receiving reprints from those whose work has not been adequately covered in this review. I should like to thank Ms Carmen Hermoso for her help with typing and Mr Jos6 Maria Gal~n for his good artwork with the figures. During the writing of this review, our laboratory was financed by grants from Plan Nacional project number (BIO 92-0715), DGICYT project number PB90-0177 and an institutional grant from Fundaci6n Ram6n Areces.