key: cord-0906941-emnpb2u9 authors: Laitinen, Olli H.; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P.; Flodström‐Tullberg, Malin title: Enteroviral proteases: structure, host interactions and pathogenicity date: 2016-05-04 journal: Rev Med Virol DOI: 10.1002/rmv.1883 sha: 20ec367d459abe3e88b7db18a359f087c37bd90e doc_id: 906941 cord_uid: emnpb2u9 Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro), are important mediators of pathology. These proteases perform the post‐translational proteolytic processing of the viral polyprotein, but they also cleave several host‐cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus‐associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro)‐mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus‐induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus‐associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease‐specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. Enterovirus infections are among the most common types of virus infections in humans. The majority of infections are subclinical, but occasionally, they cause diseases such as the common cold, hand-foot-and-mouth disease (HFMD), myocarditis meningitis, otitis media, neonatal sepsis, pancreatitis, poliomyelitis and sinusitis [1, 2] . In addition, enterovirus infections have been associated with inflammatory diseases, such as type 1 diabetes, asthma and allergies [1, 3] . Our understanding of the complex processes leading to these different disorders is limited, and a better knowledge of how these viruses interact with the host is essential for the discovery of protein; 3D pol , enteroviral RNA-dependent RNA polymerase; 3B, uridylylated-VPg; EV, extracellular vesicle; EV71, enterovirus 71; EV68, enterovirus 68; CVB, coxsackievirus; SRF, serum response factor; miRNA, micro RNA; HRV, human rhinovirus; eIF4G, eukaryotic translation initiation factor 4 gamma 1; IRES, internal ribosome entry site; CRB, cAMP response element-binding protein; Oct-1, octamer binding transcription factor 1; NLS, nuclear localization signal; NPC, nuclear pore complex; SRp20, cellular splicing factor; PCBP, cellular RNA-binding protein poly(rC)-binding protein; dsRNA, double stranded RNA; IFIH1, interferon induced with helicase C domain 1; TLR, toll-like receptor; RIG-I, retinoic acid-inducible gene I; MAVS, mitochondrial antiviral-signalling protein 1; SCARB2, scavenger receptor B2; TRIF, TIR-domain-containing adapter-inducing interferon-B; ISG, interferon-stimulated gene; G3BP1, Ras GTPase-activating protein-binding protein. disease-causing mechanisms and the identification of targets for the development of therapeutic measures. All enteroviruses encode two proteases, 2A (2A pro ) and 3C (3C pro ), which are essential for the cleavage of the viral polyprotein into structural-and nonstructural proteins. These proteases can also cleave host-cell proteins, and cellular targets already identified include transcription factors, proteins controlling nuclear import/export, mitochondriaassociated proteins, pattern recognition receptors (PRRs) and other proteins, many of which are involved in the activation of the host immune response [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] . The cleavage of host-cell proteins may contribute to pathology [14] [15] [16] , and a better insight into the target specificities of the enteroviral proteases, coupled with information on how protein cleavages affect the biological functions of the cell, is likely to reveal novel disease mechanisms as well as identify ways to treat and prevent enterovirus-mediated diseases. The molecular characteristics, such as the nature of replication, morphology and physiochemical properties of the virion define the genus Enterovirus. The genus belongs to the family of Picornaviridae, under the order of Picornavirales, and the genus is divided into twelve species: Enterovirus A-H, J and Rhinovirus A-C [17] . The enterovirus virion contains a single positive strand RNA genome with a length of around 7.5 kb. The genome is densely packed into an icosahedral capsid, which is composed of 60 copies of four separate viral capsid proteins (VP1-VP4). Upon infection, the capsid undergoes structural changes, causing the release of the viral genome into the cytoplasm where it undergoes translation by the translation machinery of the host. The enterovirus genome encodes a single open reading frame, resulting in translation of all viral proteins as a single polyprotein. Enteroviruses use several types of cell-surface molecules for binding and initiating their entry into cells ( Figure 1 ). The majority of the known enterovirus receptors belong to the immunoglobulin superfamily (IgSF) [18] , and more specifically, the type I transmembrane glycoproteins. They include the intracellular adhesion molecule (ICAM-1) [19] , the coxsackievirus-adenovirus receptor (CAR) [20] and the poliovirus receptor (PVR) [21] . Non-IgSF type receptors include decay accelerating factor (DAF), the low-density lipoprotein receptor (LDL-R), scavenger receptor B2 (SCARB2) and integrins [22] [23] [24] [25] [26] . The tissue and cell distribution of virus receptors is an important determinant for virus tropism. Polioviruses primarily infect human gastrointestinal lymphoid tissues, such as tonsils and Peyer's patches expressing the PVR [27, 28] . If the virus spreads to the circulation and, thereafter, to the central nervous system, neuronal cells expressing PVR can become infected, resulting in muscle weakness and paralysis. Through their attachment to the cell-surface receptors, enteroviruses gain access into the cell via endocytotic pathways. Routes of entry depend on the species of the virus and the cell type. The caveolae- [29] and the clathrin-dependent pathways [30] , as well as other internalization routes [31] , have been described as possible entry mechanisms. The presence of a receptor on the cell surface is, however, not the only determinant for cellular permissiveness. The virus may enter the cell but fail to replicate if, for example, there is a lack of endogenous cellular proteins required for viral propagation. An example of such an endogenous protein is the polypyrimidine tract-binding protein (PTB) [32] . Alternatively, the receptor-expressing cell can enter an antiviral state and thereby, may not be permissive to infection (reviewed in [33] ). Therefore, the dependence on various cellular factors makes host susceptibility and permissiveness to infection a multifaceted and complex phenomenon. After endocytosis, the virus particle undergoes structural changes, resulting in the uncoating of the viral genome and engagement of the capsid proteins with the endosomal membrane, presumably via the VP1 N-terminus. This allows the delivery of viral RNA with a 5′-linked VPg protein [34] and a 3′-polyadenylated tract [35] into the cytosol, where it is translated by the host ribosomes into the viral polyprotein. The polyprotein encoded by a single open reading frame is divided into three regions, P1-P3 ( Figure 1 ). The P1 region contains four structural proteins (VP1-VP4), whereas the P2 and P3 regions together contain seven non-structural proteins (2A-2C and 3A-3D), which are required in the different stages of the viruses' replication cycle. The proteolytic processing of the polyprotein into separate proteins is already initiated during translation by the viral proteases 2A pro and 3C pro [36] [37] [38] . The P1 region, encoding the structural proteins of the capsid, is the first one to be translated, followed by the P2 region, which contains three non-structural proteins (2A, 2B and 2C). During the translation of the P2 region, as 2A pro is translated first, 2A pro makes an in cis cleavage, separating itself and the P2 region from the P1 region before the full polyprotein has been translated. Translation continues through the P3 region, and this region includes the second protease, 3C pro , which is responsible for eight out of the 10 cleavages of the viral polyprotein. The cleavage carried out by the two proteases give rise to all of the non-structural proteins, with several precursor proteins, and three structural proteins: VP1, VP3 and VP0. VP0 is further cleaved into VP2 and VP4 by an unknown mechanism [39] , which may entail an RNA-mediated autocatalytic reaction during the encapsidation process [40] . Viral replication takes place in the proximity of membranous vesicles, derived partly from the endoplasmic reticulum [41] . The positive strand RNA is transcribed by the virally encoded polymerase 3D pol into a complementary negative strand RNA. The RNA synthesis is primed by uridylylated-VPg (3B), which is associated with the replication complex and recruited to the 3′ end of the negative strand viral genome to initiate RNA synthesis [42] . The negative strand RNA then serves as a template for the transcription of the positive strand RNA genome. Multiple positive-strand RNAs can be synthesized from a single negative-strand template, making positive-sense RNA abundant and directly available for translation, synthesis of additional negativesense RNA and encapsidation [43] (Figure 1 ). The accumulation of newly synthesized viral RNA and structural proteins leads to packaging of the viral genome into the capsids, thus forming new viral progeny [44] . Surprisingly, very little is known about the encapsidation process, but some studies have indicated that the process of virus assembly is coupled to RNA synthesis [45] on the surface of cytoplasmic membranes [46] . The classical view of enterovirus release is that it occurs by cell lysis. Intriguingly, new observations challenge this model as virus-containing extracellular vesicles shed by the host cells could potentially disseminate the infection [47, 48] . Persistent enterovirus infections without evident cytopathic effect in tissues and cell models have also been reported [49] [50] [51] , supporting this recently described nonlytic model of virus release. The most well-known enteroviral disease is poliomyelitis, which is caused by three different poliovirus serotypes. Poliomyelitis has been virtually eradicated in developed countries, but recently, two other enteroviruses, enterovirus 71 (EV71) and enterovirus 68 (EV68), have been demonstrated to cause an acute flaccid paralysis resembling poliomyelitis [52] [53] [54] [55] . Moreover, EV71 and coxsackievirus A6, A10 and A16 can cause HFMD [56] . Other enteroviruses, coxsackieviruses (CVBs) in particular, have been associated with acute myocarditis and the later development of dilated cardiomyopathy [14, 15, 57, 58] . Diseases related to enterovirus infections may result either from an acute infection or only appear after the acute phase is over. This indicates that there may be different mechanisms contributing to tissue pathology. Acute infections are typically associated with local inflammation (e.g. the common cold, otitis, pancreatitis and hepatitis) and are cleared relatively rapidly by the immune system. In contrast, conditions like dilated cardiomyopathy and post-polio syndrome are more likely to result from infections that have not been completely cleared and have entered a persistent infection phase. Although poliomyelitis caused by poliovirus is the most studied enterovirus-associated disease, surprisingly little is known about the disease mechanisms [59] . Even less is known on how most other enteroviruses cause disease (e.g. EV71 and EV68). An exception, however, is CVB-induced myocarditis and the subsequent development of chronic dilated myopathy, the latter a severe condition that usually leads to heart failure [15, 58] . During the acute phase of the infection, the virus-encoded protease 2A pro cleaves the cellular protein dystrophin, which leads to sarcolemmal disruption and reduction in myocyte contractility [14, 57] . In their recent publication, Matthew et al. postulated a more detailed molecular mechanism for the damage caused by the infection, namely that the C-terminal 2A pro cleavage product is retained in the sarcoglycan complex. This in turn decouples actin from the sarcolemma and subsequently prevents the recovery of the full-length dystrophin at the sarcolemmal membrane [16] . A further contribution to impaired cardiac function is the 2A pro -mediated cleavage of the transcription factor serum response factor (SRF) [60] . SRF is normally highly expressed in heart muscle cells and contributes to the regulation and expression of heart tissue-specific genes, including contractile and regulatory proteins as well as miRNAs controlling specific heart cell functions [61] . The 2A pro breaks the transactivation domain of SRF and thereby diminishes the expression of genes regulated by this transcription factor [60] . Coxsackieviruses have been shown to cause persistent infection of the heart both in animal models [62, 63] and humans [64] . Characteristic of other persistent CVB infections, they also contain deletions of varying size in their 5′ end [64] . The persistent infection may lead to a chronic immune response and also possibly autoimmune responses as exemplified by antibody responses to cardiac antigens such as cardiac myosin and troponin I [15] . The chronic inflammation is likely to contribute further to cardiac dysfunction. The enterovirus proteases 2A pro and 3C pro are multifunctional cysteine proteases, belonging to the chymotrypsin-related endopeptidase protease family [65] (MEROPS 2A pro : C03.020 and 3C pro : C03.011). When comparing 2A pro to 3C pro , a primary sequence alignment of the consensus sequences shows only~20% identity, even though the two proteases have strikingly similar tertiary structures ( Figure 2 ). Among the different species of enteroviruses, the proteases share approximately 50-75% sequence identity, the rhinoviruses being the most divergent group with around 35-55% identity with the other species ( Figure 3 ). The amino acid residues of the catalytic triad are fully conserved throughout the Enterovirus genus. In addition, the amino acid residues surrounding the catalytic residues are more conserved when compared to the rest of the protein, which is indicative of similarities in the mechanisms involving sequence specificity and cleavage among the enteroviral proteases. The tertiary structures of both of the proteases are composed of two separate domains. In the case of 2A pro , the two domains include a six-stranded antiparallel β-sheet barrel and a β-sheet pile packed on its side ( Figure 2 ). The tertiary structure of 3C pro dues, are highlighted with arrows underneath the sequences. The secondary structure elements are shown above the alignments (cylinder = alpha-helical structure; arrow = beta-sheet structure; turns in purple; 3/10 helices in pink). Secondary structure assignment was made using DSSP [66] . Panels is a combination of two twisted β-barrels, which are packed perpendicular to each other. In both proteases, these two domains participate in the formation and positioning of the catalytic triad. The catalytic triad is composed of histidine, aspartic acid and cysteine in the case of 2A pro , and histidine, glutamic acid and cysteine in the case of 3C pro . The cysteine in the catalytic triad acts as a nucleophile in the proteolytic reaction in both proteases. Characteristic for both proteases are also the conserved ion-binding motifs that are located on the opposite side from the catalytically active site. For 2A pro , a zinc ion is located in one end of the barrel, bound by three cysteines and one histidine residue. For 3C pro , a chlorine ion is bound to an Asp-Ile-Arg stretch residing in the loop connecting the two barrels. Both monomeric and dimeric quaternary structure forms have been reported for 2A pro . Liebig et al. found that HRV2 2A pro showed a dimeric state in gel filtration analysis, while CVB4 2A pro was found to be monomeric [67] . In another study, 2A pro from HRV14 was found to be monomeric by gel filtration analysis [68] . In a study by Cai et al., EV71 2A pro was found to form a disulphidelinked dimer with a negligible monomer-monomer interface in crystal structure, but the oligomeric state in solution could not be shown [69] . Mu et al. crystallized EV71 2A pro and found a monomer in the asymmetric unit [70] . In another recent study of CVA16 2A pro , both dimeric and hexameric quaternary assemblies in the solution and in crystal were reported [71] . The hexameric form was found to dissociate to dimers with an addition of DTT, which could indicate that the hexamer is not present in the reducing intracellular environment. Both dimers and hexamers, separated by size exclusion chromatography, exhibited equally efficient proteolytic activity. It is most likely that the quaternary structure of 3C pro is monomeric because it lacks a third domain, whose importance has been shown for dimerization in related coronavirus proteases [72, 73] . This is in contrast to what has been observed when solving the crystal structure, in which 3C pro proteases assembled as dimers. For example, 3C pro from EV68 and EV93 showed a dimeric assembly in crystal structures. On the contrary, they were found to be monomeric in gel filtration and DLS experiments [74, 75] . Therefore, the dimers observed in crystals are not likely to represent the biologically relevant forms. The sequence specificity, and specifically the sequences that the 2A pro and 3C pro proteases are able to cleave (or not), has not been established or studied comprehensively. To date, the amino acid residues P 4 , P 2 , P 1 , P 1 ′ and P 2 ′ are recognized as being important determinants for the sequence specificity of enteroviral proteases (Figure 4 ) [65, 76] . For the substrate recognition of 2A pro , the most important residue is P 1 ′, which is exclusively a glycine. Following P 1 ′ in order of importance are P2, occupied mainly by threonine and asparagine; P 2 ′, occupied by proline, alanine and phenylalanine; and P 4 , occupied most frequently by leucine or threonine. For 3C pro , the residues P 1 and P 1 ′ show the least amount of variance in the substrate sequence. The preferred residues for these positions are glutamine or glutamate for P 1 , and glycine, asparagine or serine for P 1 ′. In addition, the most common residue is alanine in position P 4 and proline in position P 2 ′. The most obvious feature for determining the substrate specificity of both 2A pro and 3C pro is the strong conservation of the glycine residue in position P 1 ′ [76] , and the present understanding of which residues are important in the other positions may be revised as new information becomes available (refer to the 'Methods to Identify New Cellular Substrates for Enteroviral Proteases' and 'Cleavage Predictions Using in Silico Analysis Techniques, Bioinformatics' sections in the succeeding texts). As the protease-dependent processing of the enteroviral polyprotein is indispensable for virus replication, the viral proteases have been recognized as potential targets for antiviral intervention [79, 80] . Of the two proteases, 3C pro in particular, has been considered a compelling target, as the polyprotein has several cleavage sites specific for the protease. Many of the inhibitors that have been developed and studied are small molecule peptide mimetics that target the active site of the proteases, but other small molecular compounds have also been described [81] . Structural conservation and the commonly shared proteolytic mechanism seen between different viral proteases make it possible to develop inhibitors that have an antiviral activity towards many species in the Enterovirus genus and furthermore, occasional activity towards more distantly related viruses. Such inhibitor candidates include pyrazole compounds that target 3C pro from different enteroviruses as well as coronavirus protease homologues of 3C pro [79] , microcyclic inhibitors against enterovirus 3C pro and noro-and SARS-coronavirus 3C pro homologues [82] . Additionally, a lycorine derivative, 1-acetyllycorine, has been shown to inhibit EV71 2A pro by stabilizing a special conformation of its zinc finger motive. Similarly, it can furthermore act on the homologous zinc finger of Hepatitis C virus NS3 protease [81] . The rhinovirus 3C pro inhibitor rupintrivir [83] is also active against noroviruses [84] . To date, of all the compounds studied, only rupintrivir and its analogue AG7404 (or compound 1) [85] have progressed to clinical trials [85] [86] [87] . Their development as therapeutics for rhinovirus infection has since stalled, possibly a result of their limited activity in clinical trials [88, 89] . Recently, rupintrivir has, however, gained renewed attention as it proved to be effective against EV71, CAV16 and EV68 [90] [91] [92] [93] . These interesting and optimistic results put renewed focus on the development of antivirals that target viral proteases, and it is possible that one or several novel drug candidates may show efficacy in clinical trials and reach the market in the coming years. As mentioned in the preceding texts, the enterovirus proteases fulfil several other functions in addition to cleaving the viral polyprotein into mature viral proteins. For example, they cleave cellular proteins in order to favour viral propagation over cellular protein production. The protease 2A pro interferes with and shuts down host-cell protein synthesis through cleavage of eukaryotic translation initiation factor 4 gamma 1 (eIF4G) [8] , an essential component of the cap-dependent RNA translation machinery. As enteroviruses are lacking a 7methylguanosine cap, the cleavage of eIF4G will Hydrophilic residues are shown in green colour, and hydrophobic residues are shown in black colour. Negatively charged residues are coloured red. The lower panel logos were created using all currently available enteroviral polyprotein sequences in the Uniprot database [77] . Duplicate sequences were removed to avoid bias towards sequences with multiple entries. The logos were generated using WebLogo [78] . not affect viral protein synthesis. Instead, the enteroviruses use a highly ordered secondary structure in the 5′ end of the viral RNA called the internal ribosome entry site (IRES) to achieve the initiation of translation [94, 95] . Host-cell gene transcription is also affected by enterovirus infection. During infection, the 3CD precursor protein enters the nucleus and inhibits the transcription of cellular proteins by cleavage of the TATA box, cAMP response element-binding protein, octamer binding transcription factor 1 (Oct-1) and transcriptional activating factor p53 [9, [96] [97] [98] . Although the polymerase in 3CD contains a nuclear localization signal (NLS) [99] , a recent study showed that 2A pro -mediated proteolysis is required for the nuclear translocation of 3CD [100] . In addition to a direct cleavage of cellular proteins (Tables 1 and 2 ; for more complete list of published substrates, refer to Tables S1 and S2), the proteases can also indirectly affect cellular proteins to further promote viral replication. For example, 2A pro targets several nuclear pore complex (NPC) proteins like Nup62, -98 and -153 [114, 115] . This disrupts the NPC and results in the rearrangement of nuclear proteins into the cytoplasm, where viral replication occurs. An example of a protein that is redistributed in this process is cellular splicing factor (SRp20), which binds the cellular RNA-binding protein poly(rC)-binding protein (PCBP) and recruits ribosomes to the replicating viral RNA to promote IRES-dependent initiation of the translation [114, 116] . Thus, the relocation of cellular transcription factors is utilized to modulate both viral translation and at a later stage, the generation of a new viral RNA genome [117] [118] [119] . Infected cells have several intracellular receptors that recognize different types of viruses. The enteroviruses form a dsRNA structure during replication, and the main known receptors responsible for sensing enteroviruses are interferon induced with helicase C domain 1 (IFIH1) located in the cytoplasm and toll-like receptor 3 (TLR3) in the endosomes. IFIH1 and the closely related PRR retinoic acid-inducible gene I (RIG-I) signal via a common adaptor protein called mitochondrial (Figure 1 ) [30, 108, 120, 121] . Secreted IFNs act in an autocrine or paracrine manner to trigger the cells into entering an antiviral state by the induced expression of interferon-stimulated genes (ISGs) [120, 122] . In addition to manipulating cellular proteins to favour viral replication, enteroviruses also utilize the proteases to escape recognition by the immune system. It has been shown that both 2A pro and 3C pro cleave several proteins within the viral recognition pathway, thus inhibiting the induction of IFNs. For example, viral sensors like IFIH1 and RIG-I are targets of the proteases [4, [10] [11] [12] . In addition, TRIF and MAVS, both adaptor proteins for the two major RNA sensing pathways TLR3 and IFIH1/RIG-I, are cleaved by 3C pro and/ or 2A pro [4, 5, 13, 108] , and downstream proteins like the transcription factor IRF7 can be targeted as well [5] . It has also been shown that EV71 2A pro acts directly on the interferon receptor 1, reducing its expression and thereby impairing the efficacy of IFN as a treatment against infection [7] . Given that it has been noted that enteroviral proteases can contribute to disease pathology (e.g. cleavage of dystrophin in the heart muscle), it is possible that other enteroviral diseases are also associated with proteolytic activities of 2A pro and/or 3C pro . The identification of additional host-cell proteins that are targeted by the proteases may thus lead to the identification of novel disease mechanisms. Because enteroviruses are able to cause diverse diseases affecting different tissues and organs, it may also be of relevance to understand how these proteases act in specific tissues and cells. There are number of approaches that have been used to study the cellular targets of 2A pro and 3C pro . These include infection of cells or tissues, for example, [108, 118, [123] [124] [125] , selective overexpression of viral proteases by transfection, for example, [7, 108] , transgenic techniques [60] , a variety of in vitro assays, in which the proteases have been incubated with cell lysates, for example, [108, 118, 126] , and in silico prediction of the cleavage sites based on amino acid sequences and composition of potential target proteins [76] . To analyse whether the experimental approaches result in cleavage by enteroviral enzymes, Western blotting is a frequently used method. With Western blotting, it is possible to observe the appearance of cleavage products and/or a decrease in the concentration of the potential target proteins (e.g. Figure 5 ). However, because antibodies may not recognize the produced fragments, this analysis can be cumbersome. Transfection studies have been used to reveal the protein responsible for the effects observed in the infected cells. Nevertheless, when conducting transfection studies to overexpress a selected viral protein, a caveat may be that the function of the viral protein might be dependent on other viral proteins, for example, [100] . Also, it must be taken into account that the protease precursors could have different protein targets compared to mature proteases. The technical limitations mentioned in the preceding texts may provide an explanation for the contradictory reports in the literature. One study indicated that the 3C pro of coxsackievirus B3 can cleave MAVS [13] , while other studies suggest that this cleavage is mediated by 2A pro [4, 108] . Enterovirus infections can also activate endogenous proteases including caspases, which also cleave cellular proteins. Barral et al. [10] showed that poliovirus-induced apoptosis during the course of infection correlates with the cleavage of MDA5. This cleavage also appeared after the cells were treated with puromycin, an inducer of apoptosis. Thus, activation of endogenous proteases may result in the erroneous identification of protein targets for 2A pro and 3C pro . Another drawback in the analyses described in the preceding text is that they are all hypothesisdriven. Researchers identify a protein of interest and address whether it is affected by an enterovirus-encoded protease. The outcome is that only one or a few cellular proteins are studied at the time. In order to overcome this shortage, a proteome-wide approach was presented by Weng et al. [106] , who identified new 3C pro substrates using nuclear extracts that were treated with the 3C pro in vitro. The treated lysates were analysed with the combination of 2D electrophoresis and mass spectrometry. They identified eight novel substrates for 3C pro , out of which they analysed the cleavage of stimulation factor 64 in more detail. Newer methodologies in quantitative proteomics have recently been used to study how enteroviruses affect the host-cell proteome [124, 125] , and such methods may also be applied to identify new protease targets [127] . A potential disadvantage with these type of analyses is that they are restricted to the proteins expressed by the infected cell, and will not provide a simultaneous analysis of the whole human proteome. The sequences and structures of the viral polyproteins, as well as their identified cellular targets, may form the basis for the prediction of novel cellular protein substrates. The most comprehensive work completed to predict new cleavage sites of the enteroviral proteases 2A pro and 3C pro has been performed by Blom et al. [76] through the use of a neural network algorithm for prediction. In their study, they used a collection of known cleavage sites to teach the algorithm how to predict the potential cleavage sites. The algorithm scores amino acid sequences for potential cleavage sites based on two calculated parameters, the first being sequence specificity, and the second being surface accessibility. The algorithm is published and available as a free tool on the Internet: NetPicoRNA Server (http://www.cbs.dtu.dk/services/NetPicoRNA/). NetPicoRNA server seems, however, to underestimate the number of 2A pro cleavage targets, as for example, Nup98 is not recognized as a potential candidate, while the number of 3C pro cleavage targets may be overestimated. At the time of the publication of the server (1996), only a limited number of cellular substrates were known, which may have caused a bias to certain kind of cleavage sites. In addition, the surface accessibility prediction was not based on resolved 3D structures, but on ab initio primary sequence analysis and the amino acid compositions of the proteins. Many new substrates have been identified since 1996 (Tables 1 and 2 , Tables S1 and S3, and references therein), and a large amount of the human proteome 3D structural data is now available [128] . Indeed, by the end of year 1996, the number of the structures reported in the PDB [129] was 5915, while from 1997 to 2014, this number increased to above 93 000. Therefore, it may be worth revising both the sequence specificity, as well as the surface accessibility predictions, with an aim to develop an improved algorithm that can be useful in identifying novel substrates. Such work is ongoing in our laboratories. Enteroviruses are important human pathogens whose manifestations range from subclinical infections to severe life-threatening diseases. Hospital visits and symptomatic treatments for the severe infections are costly, and it is clear that a better understanding for the complex disease mechanisms underlying enterovirus-mediated diseases would lead to more efficacious treatments. A few disease mechanisms have been identified, and in the near future, additional ones are likely to be discovered by research teams that integrate many scientific disciplines such as bioinformatics, molecular biology, proteomics and the use of patient materials. Several pathological mechanisms may be explained by the activity of the viral proteases 2A pro and 3C pro . Some effects, such as the shutting down of host-cell protein synthesis, immune evasion, as well as the hijacking of the cellular machinery to favour virus propagation, may be common to most enteroviruses. However, especially in the persistent types of infections when the production of viral proteins, including proteases 2A pro and 3C pro , continues for months or years, the degradation of host proteins may be virus-and tissue-specific and may lead to more selective pathological processes (e.g. myocarditis and the development of dilated cardiomyopathy). Currently, there are only a limited number of studies that have addressed the role of the proteases in a tissue-specific manner. Better tools to globally identify and verify protease targets should assist in the identification of novel cellular protein substrates without limitations to particular cell types. Overall, this should provide a better understanding of how the proteases, in concert with other viral proteins, contribute to the induction of different diseases. Such information will also be of immediate importance for the development of novel drugs, including protease inhibitors, to prevent and treat diseases caused by enteroviruses. New prediction methods and proteome-wide approaches are critical for the successful completion of this goal. 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