key: cord-0766774-sneqefec
authors: Cavanagh, D.; Britton, P.
title: Coronaviruses: General Features
date: 2008-07-30
journal: Encyclopedia of Virology
DOI: 10.1016/b978-012374410-4.00370-8
sha: f665ef45c215f8be7f3805f38e062a30f4bde6b3
doc_id: 766774
cord_uid: sneqefec

Coronaviruses have the largest known RNA genomes (∼30 kb), which are of positive sense. Together with toroviruses, they are classified in the family Coronaviridae, order Nidovirales. All coronaviruses have four common proteins, three in the envelope and one associated with the genome. Assembly of virus particles occurs at internal membranes. The genes for the structural proteins are at the 3′ end of the genome. Most of the genome (∼20 kb) is gene 1, which encodes 15–16 proteins associated with RNA replication and transcription. Translation of gene 1 involves ribosomal frameshifting. Transcription is by a discontinuous process which results in a 3′ co-terminal nested set of mRNAs, each of which has a common leader sequence transcribed from the 5′ terminus of the genome. Only the most 5′-proximal gene of each mRNA is translated. Recombination is a feature of coronavirus evolution. The outbreak of severe acute respiratory syndrome (SARS) has resulted in the discovery of more coronaviruses in humans, other mammals, and avian species, and the realization that the host range of coronaviruses is wider than previously acknowledged. Coronaviruses are associated with a wide range of diseases, including the respiratory and enteric systems, though not necessarily restricted to these, for example, some coronaviruses affect the central nervous system, kidneys, and gonads. The most widely used coronavirus vaccine (billions of doses annually) is against infectious bronchitis virus, which affects chickens.

Coronaviruses are known to cause disease in humans, other mammals, and birds. They cause major economic loss, sometimes associated with high mortality, in neonates of some domestic species (e.g., chickens, pigs). In humans, they are responsible for respiratory and enteric diseases. Coronaviruses do not necessarily observe species barriers, as illustrated most graphically by the spread of severe acute respiratory syndrome (SARS) coronavirus among wild animals and to man, with lethal consequences. As a group, coronaviruses are not limited to particular organs; target tissues include the nervous system, immune system, kidney, and reproductive tract in addition to many parts of the respiratory and enteric systems. A great advance in recent years has been the development of systems ('infectious clones') for modifying the genomes of coronaviruses to study all aspects of coronavirus replication, and for the development of new vaccines.

The genus Coronavirus together with the genus Torovirus form the family Coronaviridae ; members of these two genera are similar morphologically. The Coronaviridae, Arteriviridae, and Roniviridae are within the order Nidovirales. Members of this order have a similar genome organization and produce a nested set of subgenomic mRNAs (nidus, Latin for nest). To date, coronaviruses have been placed into one of three groups (Table 1) . Initially, this was on the basis of serological relationships which subsequently have been supported by gene sequencing.

Virions have a buoyant density of approximately 1.18 g ml -1 in sucrose. Being enveloped viruses (Figure 1(a) ), they are destroyed by organic solvents such as ether and chloroform.

All coronaviruses have four structural proteins in common (Figure 1(b) ): a large surface glycoprotein (S; c. 1150-1450 amino acids); a small envelope protein (E; c. 100 amino acids, present in very small amounts in virions); integral membrane glycoprotein (M; c. 250 amino acids); and a phosphorylated nucleocapsid protein (N; c. 500 amino acids). Group 2a viruses have an additional structural glycoprotein, the hemagglutinin-esterase protein (HE; c. 425 amino acids). This is not essential for replication in vitro and may affect tropism in vivo.

Virions are c. 120 nm in diameter, although they can be up to twice that size, and the ring of S protein spikes is approximately 20 nm deep. When present, the HE protein forms a layer 5-10 nm deep. In some species, the S protein is cleaved into two subunits, the N-terminal S1 fragment being slightly smaller than the C-terminal S2 sequence. The S protein is anchored in the envelope by a transmembrane region near the C-terminus of S2. The functional S protein is highly glycosylated and exists as a trimer. The bulbous outer part of the mature S protein is formed largely by S1 while the stalk is formed largely by S2, having a coiled-coil structure. S1 is the most variable part of the S protein; some serotypes of IBV differ from one another by 40% of S1 amino acids. S1 is the major inducer of protective immune responses. Variation in the S1 protein enables one strain of virus to avoid immunity induced by another strain of the same species.

The M glycoprotein is the most abundant protein in virions. In most cases, only a small part (20 amino acids) at the N-terminus protrudes at the surface of the virus. There are three membrane-spanning segments and the C-terminal half of the M protein is within the lumen of the virus. In transmissible gastroenteritis virus (TGEV), a proportion of M molecules have four membranespanning segments, resulting in the C-terminus also being exposed on the outer surface of the virus (M 0 in Figure 1(b) ). The E protein is anchored in the membrane by a sequence near its N-terminus.

Coronaviruses have the largest known RNA genomes, which comprise 28-32 kb of positive sense, single-stranded RNA. The overall genome organization is being 5 0 UTRpolymerase gene-structural protein genes-3 0 UTR, where the UTRs are untranslated regions ( Figure 2 ). The first 60-90 nucleotides at the 5 0 end form a leader sequence. The structural protein genes are in the same order in all coronaviruses: (HE)-S-E-M-N. Interspersed among these genes are one or more gene (depending on the species; SARS-CoV has four) that encode small proteins of unknown function. Some of these genes encode two or three proteins. In some cases (e.g., gene 3 of IBV and gene 

and second open reading frame (ORF), respectively, is effected by the preceding ORFs acting as internal ribosome entry sites. The proteins encoded by these small ORFs are mostly not required for replication in vitro; some of them might function as antagonists of innate immune responses, though this has not yet been demonstrated.

Following entry into a cell and the release of the virus ribonucleoprotein (genome surrounded by the N protein) into the cytoplasm, ribosomes translate gene 1, which is approximately 20 kb, into two polyproteins (pp1a and pp1ab). These are cleaved by gene 1-encoded proteases, to generate 15 or 16 proteins (Figure 3) . Translation of ORF 1b involves ribosomal frameshifting, which has two elements, a slippery site followed by an RNA pseudoknot. At the slippery site (UUUAAAC in IBV), the ribosome slips one nucleotide backward and then moves forward, this time in a -1 frame compared with translation ORF 1a, resulting in the synthesis polyprotein 1ab.

Proteins, including the RNA-dependent RNA polymerase, from gene 1 associate to form the replicase complex, which is membrane associated. Coronavirus subgenomic mRNAs are generated by a discontinuous process. At the beginning of each gene is a common sequence (CUUAACAA in the case of IBV) called a transcription regulatory sequence (TRS). It is believed that when the polymerase producing the nascent negative sense RNA, reaches a TRS, RNA synthesis is attenuated, followed by continuation at the 5 0 end of genomic RNA. This results in the addition of a negative copy of the leader sequence to the negative-sense RNA, resulting in a negative-sense copy of an sg mRNA. Of course, progress of the polymerase is not always halted at a TRS. Rather, it sometimes continues, producing a nested set of negativesense sg mRNAs. These are the templates for the generation of the positive-sense sg mRNAs ( Figure 2 ). The amount of each sg mRNA does not necessarily decrease in a linear fashion; the efficiency of termination by a TRS is dependent on adjacent sequences, which are different for each gene. The leader sequence is found at the very 5 0 end of the genomic RNA and at the 5 0 ends of each sg mRNA.

The N-terminal (S1) part of the S protein mediates that mediates attachment to cells. It is a determinant of host species specificity and, in some cases, pathogenicity, by determining susceptible cell range (tissue tropism) within a host. The C-terminal S2 part triggers fusion of the virus envelope with cell membranes (plasma membrane or endosomal membranes), which can occur at neutral or slightly acidic pH, depending on species or even strain. The virus glycoproteins (S, M, and HE, when present) are synthesized at the endoplasmic reticulum. Both subunits The leader sequence, represented by a gray box, is at the 5 0 end of the genomic RNA and at the 5 0 ends of the sg mRNAs. The genomic RNA is translated to produce two polyproteins, pp1a and pp1ab, that are cleaved by virus-encoded proteases to produce the replicase proteins. The structural proteins, S, E, M, and E, and the accessory proteins, 3a, 3b, 5a, and 5b, produced from IBV genes 3 and 5, respectively, are translated from the sg mRNAs. The proteins produced by the sg mRNAs are represented by lines below the corresponding sg mRNA. All of the sg mRNAs, except the smallest species, are polycistronic but only produce a protein from the 5 0 -most gene. The ribosome frameshift (RFS) region, denoted as a black circle on the genomic RNA, directs the -1 frameshift event for the synthesis of pp1ab. Translation of the genomic RNA results in the production of pp1a. However, the translating ribosomes undergo the -1 frameshift about 30% of the time resulting in pp1ab. The 5 0 and 3 0 UTR sequences are represented as single lines downstream of the leader and N gene sequences, respectively. of the S protein are multiply glycosylated, while the M protein has one or two glycans close to its N-terminus. Interestingly, glycosylation of the M protein can be either N-or O-linked, depending on the type of coronavirus, although experiments using reverse genetics showed that conversion of an O-linked glycosylated M protein to an N-linked version had no effect on virus growth.

Early and late in infection, formation of virus particles can occur in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and endoplasmic reticulum, but most assembly occurs in the Golgi membranes. The M protein is not transported to the plasma membrane; its location at internal membranes determines the sites of virus particle formation. It interacts with the N protein (as part of the RNP) and C-terminal part of the S protein, retaining some, though not all, of the S protein at internal membranes. The E protein is essential for virus particle formation, though it is not known how it functions. It has a sequence that determines its accumulation at internal membranes, and its interaction with the M protein. The latter interacts with the C-terminus of the S protein, retaining some of it at internal membranes, and with the N protein (itself part of the ribonucleoprotein structure), enabling the formation of virus particles with spikes.

Following infection of a susceptible cell, the coronavirus genomic RNA is released from the virion into the cytoplasm and immediately recognized as an mRNA for the translation of the replicase pp1a and pp1ab proteins. These proteins are cleaved by ORF1a-encoded proteases, after which they become part of replicase complexes for the synthesis of either complete negative-sense copies of the genomic RNA or negative-sense copies of the sg mRNAs. The negative-sense RNAs are used as templates for the synthesis of genomic RNA and sg mRNAs (Figure 2) . Following synthesis of the sg mRNAs, the structural proteins are produced for the assembly and encapsidation of the de novo-synthesized genomic RNA, resulting in the release of new infectious coronavirus virions. The release of new virions starts 3-4 h after the initial infection. As indicated above, the synthesis of the sg mRNAs is the result of a discontinuous process in which the synthesis of a negative-sense copy of an sg mRNA is completed by the addition of the negative-sense leader sequence by a recombination mechanism. If a cell is infected with two related coronaviruses, the polymerase may swap between two RNA templates, in a similar way to addition of the leader sequence. This 'copy-choice' mechanism of genetic recombination results in a chimeric RNA. Such RNAs may give rise to new viruses with modified genomes with a capacity to infect a different cell and, in some cases, new host species.

Phylogenetic analyses of the structural proteins have resulted in the grouping of coronavirus species in accordance with earlier antigenic groups (Table 1 and Figure 4 ). Figure 3 Organization of the coronavirus replicase gene products. Translation of the coronavirus replicase ORF 1a and ORF 1b sequences results in pp1a and pp1ab; the latter is a C-terminal extension of pp1a, following a programmed -1 frameshift event (see legend to Figure 2 ). The two polyproteins are proteolytically cleaved into 10 (pp1a; nsp1-11) and 16 (pp1ab; nsp1-16) products by the papain-like proteinases (PL1 pro and PL2 pro ) and the 3C-like (3CL pro ) proteinase. The PL pro proteinases cleave at the sites indicated with a black triangle and the 3CL pro proteinase cleaves at the sites indicated with a gray triangle. The nsp11 product of pp1a is produced as a result of the ribosomes terminating at the ORF 1a translational termination codon, a -1 frameshift results in the generation of nsp12, part of the pp1ab replicase gene product. Various domains have been identified within some of the replicase products: Ac is a conserved acidic domain; X ¼ ADP-ribose 1 0 -phosphatase (ADRP) domain; PL1 and PL2 the two papain-like proteinases; Y is a conserved domain; TM1, TM2, and TM3 are conserved putative transmembrane domains; 3CL ¼ 3CL pro domain; RdRp, RNA-dependent RNA polymerase domain; HEL, helicase domain; ExoN, exonuclease domain; NendoU, uridylate-specific endoribonuclease domain; MT, 2 0 -O-ribose methyltransferase domain. nsp's 7-9 contain RNA-binding domains (RBDs).

Members of subgroups have higher amino acid sequence identities to each other (60%) than to members of another group in the same group (with which they share 40% identity). Comparing one group with another, protein sequence identities are generally in the range 25-35%. Unlike other members of group 2, SARS-CoV does not have an HE glycoprotein. Phylogenetic analysis using all the encoded proteins indicates that recombination has been a feature of coronavirus evolution. For example, some group 1a viruses are clearly recombinants between a feline and canine group 1 coronavirus.

Probably all coronaviruses replicate in epithelial cells of the respiratory and/or enteric tracts, though not necessarily producing clinical damage at those sites. Avian IBV not only causes respiratory disease but can also damage gonads in both females and males, and causes serious kidney disease (dependent on the strain of virus, and to some extent on the breed of chicken). IBV is able to replicate at virtually every epithelial surface in the host. Some coronaviruses have their most profound effect in the alimentary tract (e.g., porcine TGEV causes 90% mortality in neonatal pigs). Human coronaviruses are known to be associated with enteric and respiratory diseases (e.g., diarrhea), in addition to respiratory disease. SARS-CoV was also associated with diarrhea in humans, in addition to serious lung disease. Other coronaviruses, for example, MHV and porcine HEV, spread to cells of the central nervous system, producing disease, for example, acute or chronic demyelination in the case of MHV.

Coronavirus replication and disease are not necessarily restricted to a single host species. Canine enteric CoV and feline CoV can replicate and cause disease in pigs; these two viruses have proteins with very high amino acid identity to those of porcine TGEV. Canine respiratory CoV has proteins, including the S protein (which is the attachment protein and a determinant of host range), with very high amino acid identity (95%) to other group 2 viruses Hu CoV-OC43 and BCoV. This raises the possibility of co-infection in these hosts. Bovine CoV causes enteritis in turkeys following experimental oral infection. There is evidence that pheasant CoV can infect chickens, and IBV infect teal (a duck), though without causing disease. The most dramatic demonstration that coronaviruses can have a wide host range was provided by SARS-CoV. This may have had its origin in bats, was transferred to various other species (e.g., civet cat) that were captured for trade, and then caused lethal disease in humans.

Persistent infections in vivo are well known for MHV, and less well known for other coronaviruses (e.g., IBV). Following infection of very young chickens, IBV is reexcreted when hens start to lay eggs. The trigger for release is probably the stress of coming into lay.

The S protein is a determinant of both tissue tropism within a host and host range. This has been elegantly demonstrated by genetic manipulation of the genome of MHV, which is unable to attach to feline cells. Replacement of the MHV S protein gene with that of CoV from feline coronavirus resulted in a recombinant virus that was able to attach, and subsequently replicate in, feline cells. However, other proteins can also affect pathogenicity. Research with genetically modified coronaviruses, using targeted recombination or 'infectious clones', has shown that modifications to proteins encoded in ORF1 and the small genes interspersed among the structural protein genes, result in attenuation of pathogenicity. Although the roles of these 'accessory proteins' are not known, this may offer a route to the development of a new generation of live vaccines. Currently, the most widely used prophylactics for control of IBV in chickens include killed vaccines and live vaccines attenuated by passage in embryonated eggs. However, disease control is complicated by extensive variation in the S1 protein which is the inducer of protective immunity. Figure 4 Phylogenetic relationship of aligned coronavirus-derived nucleoprotein amino acid sequences. The complete N protein sequences represent coronaviruses from each of the three groups ( Table 1) . The tree is unrooted and the three main coronavirus groups, 1-3, are highlighted as dark gray ellipsoids. Groups 1 and 2 are divided into two subgroups, a and b, representing some divergence of the sequences within their corresponding groups. Similar relationships are observed when comparing other structural proteins and replicase-derived proteins.

Coronaviruses (CoVs) were first identified during the 1960s by using electron microscopy to visualize the distinctive spike glycoprotein projections on the surface of enveloped virus particles. It was quickly recognized that CoV infections are quite common, and that they are responsible for seasonal or local epidemics of respiratory and gastrointestinal disease in a variety of animals. CoVs have been named according to the species from which they were isolated and the disease associated with the viral infection. Avian infectious bronchitis virus (IBV) infects chickens, causing respiratory infection, decreased egg production, and mortality in young birds. Bovine coronavirus (BCoV) causes respiratory and gastrointestinal disease in cattle. Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) cause gastroenteritis in pigs. These CoV infections can be fatal in young animals. Feline infectious peritonitis virus (FIPV) and canine coronavirus (CCoV) can cause severe disease in cats and dogs. Depending on the strain of the virus and the site of infection, the murine CoV mouse hepatitis virus (MHV) can cause hepatitis or a demyelinating disease similar to multiple sclerosis. CoVs also infect humans. Human coronaviruses (HCoVs) 229e and OC43 are detected worldwide and are estimated to be responsible for 5-30% of common colds and mild gastroenteritis. Interestingly, HCoV-OC43 and BCoV share considerable sequence similarity, indicating a likely transmission across species (either from cows to humans or vice versa) and then adaptation of the virus to its host. In contrast to the relatively mild infections caused by HCoV-229e and HCoV-OC43, the CoV responsible for severe acute respiratory syndrome (SARS-CoV) causes atypical pneumonia with a 10% mortality rate. Two additional HCoVs, HCoV-NL63 and HCoV-HKU1, have been recently identified using molecular methods and are associated with upper and lower respiratory tract infections in children, and elderly

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