The picornavirus family is one of the largest virus families known, and is composed of nine genera: the rhinovirus, hepatovirus, teschovirus, erbovirus, cardiovirus, parechovirus, kobuvirus, aphthovirus, and enterovirus. These genera consist of several pathogens, implicated in an extensive range of clinical manifestations that affect both humans and animals. Although often mild and self-limiting, picornaviruses could also be involved in more serious conditions, which are proven to be life threatening.
A picornavirus belongs to Picornaviridae family. Picornavirions are small, non-enveloped, and spherical in shape with a diameter of approximately 30 nm. They contain a single stranded, positive sense RNA genome that is around 7.2 and 9.0 kilobases long. The genetic information that exists in the single-stranded positive sense RNA genome is expressed as a single protein of approximately 2000 amino acids1. This primary product of protein synthesis, designated the polyprotein, is properly cleaved into the mature viral proteins by proteinases which are found within it. Despite the fact that most infections in man are de-escalating mildly or asymptomatically, picornaviruses can also be responsible for severe, potentially life-threatening diseases. Even though currently there are drugs that are tested through clinical trials, and efforts to develop an antiviral that is effective in treating picornavirus-associated diseases are ongoing2, no therapy has been approved for the treatment of picornavirus infections.
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Several important steps in the picornaviral replication cycle, involving structural as well as nonstructural proteins have been identified, and the following discussion will reveal their role in the replication of the virus.
The icosahedrally shaped capsids are formed from 60 protomers, where each of those is composed of four structural proteins: VP1 (viral protein 1), VP2, VP3, and VP4. The shell of the virus is created from VP1, VP2, and VP3. Different varieties within these capsid proteins lead to different antigenic serotypes in the picornavirus family. On the other hand, VP4 is located in the inside part of the surface and serves in attaching the capsid to the RNA genome. Uncoating of the virus occurs upon destabilization of VP4. The surface of the virion shows a fivefold axis of symmetry, surrounded by a deep depression or canyon3.
Covalently attached to its 50-end, the viral genome has a small protein called VPg, which is involved in the initiation of viral RNA replication.
The genomic RNA has a highly structured 50 non-coding region that contains the interna ribosome entry site (IRES) and a short 30 non-coding region, followed by a poly(A) tract. Of course some specific picornaviruses (aphtovirus and some cardiovirus) genomes contain an extended internal poly(C) tract within the 50 UTR. The non-coding regions at both ends play a role in replication efficiency, virus infectivity, tissue tropism and other regulatory activities during viral replication and translation4.
The coding region of the viral genome contains both non-structural and structural viral proteins which are divided into three primary precursor molecules (P1, P2, and P3). The structural proteins which include the viral capsid are originated from the P1 portion of the polyprotein, while the non-structural proteins are encoded by the P2 and the P3 regions. These non-structural proteins consist of two proteases (2A and 3C), one polymerase (3D), one ATPase (2C) and four other proteins. Even if they are cleaved or act as a precursor, it seems that they are involved in viral replication. In addition to these proteins, the aphtoviruses and cardioviruses also code for an L protein at the N-terminus of their polyproteins. During the binding of the receptor, the capsid of the virus is destabilized and the release of VP4 occurs5. These procedures are executed after the viral RNA enters the cytoplasm of the host cell. The viral genome plays the role of a template for both viral protein translation and RNA replication (Fig. 2). RNA replication occurs through the cooperation of cellular membranes, and new positive-strand RNA genomes are synthesized through a negative-strand intermediate6. These RNA strands are gathered and packaged into new viral structural proteins, and work together to form new viral particles, which are then released from the host cell.
Proteolytic processing of the viral polyprotein into intermediate precursors and mature proteins could be mediated by three proteases which exist in the L, 2A and 3C proteins (Fig. 2.3). Nevertheless, most picornaviruses use only one or two proteases. Proteolytic cleavage at the conserved interdomain junctions of the polyprotein of all picornaviruses is mostly performed by the 3C section, which contains a chymotrypsin-like cysteine protease. Aphthoviruses, erboviruses, cardioviruses, kobuviruses, teschoviruses and unclassified porcine enterovirus 8 encode L proteins, also reveal that some of them are proteolytically active. This is proved from the fact that L protein of Foot-and-Mouth Disease Virus (FMDV), cleaves at its own C terminus7. Despite the fact that erbovirus' L protein also has an autocatalytic activity, has limited sequence similarities with the FMDV L protein. Regarding the comparison between picornaviruses with different L proteins, kobuviruses cardioviruses present no significant homology to other picornavirus L proteins and do not have autocatalytic activity. L proteins seem to be released from the polyprotein by the viral 3C protease8.
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Even if 2A protein is encoded from every picornavirus, some structurally and evolutionary not related forms of this protein seem to exist. Among Picornaviruses, enterovirus' and rhinovirus' 2A proteins only, seem to be capable of having an active a chymotrypsin-like protease, which processes the VP1-2A junction9. In these viruses the 2A-2B junction is cleaved by the 3C protease. In this situation it seems that the opposite occurs, with the release of the N-terminus of the unrelated 2A protein of cardioviruses and aphthoviruses, which is performed by the 3C protease, whereas the release of the 2A C-terminus is mediated by a unique co-translational peptide scission event controlled by the 2A protein10.
Picornavirus 3C and 2A proteases are known to target cellular substrates except from their role in the maturation of the viral polyprotein. It is generally believed that the translation inhibition in Picornavirus-infected cells is induced by 3C- and 2A-mediated cleavage of host proteins involved in transcription, translation, and cytoskeletal integrity11. A number of RNA polymerase transcription factors including Octamer binding protein (Oct-1), TATA-binding protein (TBP) and cyclic AMP-responsive element binding protein (CREB) are cleaved by the 3C protease. TATA-binding associated factor 110 (TAF 110), transcription factor IIIC (TFIIIC), cytoskeletal protein MAP-4 (microtubule-associated protein 4), and the polyA-binding protein (PABP), are also cleaved by the 3C protease12. The aforementioned protease in the case of FMDV induces proteolytic cleavage of host cell histone H3 and cleaves the translation initiation factor eIF4G. On the other hand, coxsackieviruses translation initiation factor eIF4G for example, are not a substrate of the HAV 3C protease13.
The picornavirus 2A protease is also known to cleave proteins that are involved in host cell transcription, (TBP), the cytoskeletal protein dystrophin, PABP and the translation initiation factor eIF4G. It is possible that the shut-off of host cell transcription is greatly involved in the replication of picornaviruses14, 15.
Still, the exact mechanisms by which picornaviruses mediate nearly complete translation inhibition in host cells remain mostly indefinable. For example in PV-infected Hela cells, partial translation inhibition was originally thought to result from cleavage of eIF4GI by the viral 2A protease, although more recently was discovered that also cellular proteases activated during infection have their part in eIF4GI cleavage16. Still, cleavage of eIF4GI seems to be only partially responsible for the translation shutoff. This indicates the fact that additional events are required to result in complete host cell translation shutoff. An additional factor could be the cleavage of PABP, given that both the 2A protease and 3C protease cleave PABP during enterovirus infection. Additionally to its proteolytic activity, the ability to specifically bind viral RNA is unique to picornaviral protease 3C (and its precursors). For PV, HRV and HAV secondary structures formed at the 5´ end of their genomes were identified as specific RNA targets for binding through viral proteases. In the case of PV, it was proposed that the stable 3C precursor known as 3CD (see also below) is involved in the switch from translation to RNA synthesis by binding to the 5´ end of the viral genome17.
Polyprotein precursors or processing intermediates often have functions in replication that are different from those of the mature cleavage products. An example of a molecule exhibiting such differential functions is the 3CD product of picornaviruses, containing RNA binding and protease activities that reside in its 3C moiety and the silent RNA-dependent RNA polymerase in the 3D domain. In viral RNA replication, 3CD forms a ternary ribonucleoprotein (RNP) complex with the 5'-terminal sequences of genomic RNA and a cellular RNA-binding protein termed poly(rC)-binding protein 2 (PCBP2) or the viral protein 3AB, but also exhibits protease activity towards all 3C cleavage sites in the polyprotein18. Moreover, biochemical studies on PV 3C and 3CD enzymes showed that processing of the viral capsid precursor is in fact more efficiently mediated by 3CD than by 3C. 3CD is also able to trans-cleave 3CD molecules more efficiently than 3C does, and it processes sites within the P3 precursor more rapidly. There were no differences found between 3C and 3CD in the processing of a nonstructural polyprotein precursor, 2C3AB19.
Thus far, the exact biochemical roles of specific 3D amino acid sequences and domains for 3CD protease activity are weakly understood. Possibly, the structural domains within the 3D portion of the 3CD help to the improved activity of this protease toward 3C cleavage sites residing in the P1 precursor. It is possible that 3C or a precursor of 3C enters the nucleus of infected cells to shut-off host cell transcription20.
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Although dissemination of 3C into the nucleus, when present at sufficiently high concentrations cannot be ruled out, another explanation seems more likely. A single basic type of nuclear localization signal (NLS) was identified in the 3D domain. Possibly, 3C enters the nucleus in the form of its precursor, 3CD, which then generates 3C by auto-proteolysis leading to cleavage of transcription factors. However, the presence of the NLS alone was not enough for nuclear entry of 3C/3CD. This self-implies that other cofactors may be required or upon PV infection additional alterations in the nuclear membrane are induced which enable successful nuclear translocation of 3C/3CD21.
Another virus-encoded protein that may regulate polyprotein processing, is the 2C protein, that is highly conserved among picornaviruses and has been concerned in a number of functions during viral replication such as uncoating, host cell membrane rearrangement, RNA replication, and encapsidation22. It is quite important to say that the exact role of 2C in these processes is not fully understood. It was demonstrated that the purified 2C protein is capable of inhibiting the activity of both the 3C and 2A proteases in PV-infected cells.
It is possible that 2C downregulates 3C activity by physically interacting with it, which was demonstrated by co-immunoprecipitation experiments. Mutations in the amphipathic helix of 2C, which was proposed to be responsible for its membrane binding properties, resulted in abnormalities in polyprotein processing of the P2 and P3 region by the 3C protease, which confirms a possible regulatory role for 2C in 3C-mediated polyprotein processing23.
Acquiring all this knowledge around the proteins of picornaviruses, was at the same time a substrate to build on strategic plans in order to find ways of treatment of different picornavirus types.
Since the genomic RNA 3Î„ noncoding region seems to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis, studies showed that complete deletions of the genomic RNA 3Î„ noncoding regions could affect the basic mechanism of replication initiation which is not firmly template specific and may rely mainly upon the proximity of newly translated viral replication proteins to the 3Î„ terminus of template RNAs within tight membranous replication complexes24.
Studies in switching amino acids in specific positions of specific proteins were also carried out. For example mutations into the 3CÎ„/3DÎ„ cleavage site in an infectious cDNA clone of poliovirus type 1 by oligonucleotide-directed mutagenesis were induced, in order to examine the role of 3CÎ„and 3DÎ„ in viral proliferation and to obtain information about the cleavage specificity of 2Apro, and observe differences or similarities to the phenotypes of the mutant viruses with that of the parental strain25.
Studies on eIF4G1 showed that one activity cleaves eIF4G1 at or very near the 2Apro cleavage site and the other activity cleaves approximately 40 residues upstream of the 2Apro cleavage site. C-terminal cleavage fragments of eIF4GI were purified from infected cells, and a new eIF4GI cleavage site was mapped to a unique site 43 amino acids upstream of the known 2Apro cleavage site. Further, eIF4GI cleavage in vivo could be blocked by addition of zVAD to PV-guanidine infections, suggesting that the same types of eIF4GI cleavage activities which are generated in PV-infected cells can also be generated in the absence of virus16.
Studies on inhibiting the replication of specific were also carried out. Pyrrolidine dithiocarbamate (PDTC) inhibits proteolytic polyprotein processing and replication of human rhinovirus by transporting metal ions into cells. It seems that PDTC also inhibits replication of two other picornaviruses: coxsackievirus B3 (CVB3), a closely related virus that belongs to the genus Enterovirus, and mengovirus, an encephalomyocarditis virus strain that belongs to the genus Cardiovirus, and that this inhibition is due to the dithiocarbamate moiety of the compound. Evidence revealed that PDTC inhibits replication of these two viruses through the disruption of the viral RNA synthesis. Furthermore, PDTC carries zinc ions into cells which play an important role in the antiviral activity mediated by PDTC. Studies showed that PDTC interferes with proteolytic processing of the polyproteins of both CVB3 and mengovirus, but that the underlying mechanism between these two viruses differs26. In CVB3-infected cells, PDTC interferes powerfully with the proteolytic activity of 3CDpro, as shown by the impaired production of the mature capsid proteins as well as the autocleavage of 3CDpro into 3Cpro and 3Dpol. In mengovirus-infected cells though, PDTC had no effect on the proteolytic production of capsid proteins or the autocleavage of 3CDpro. PDTC in the researchers surprise caused the accumulation of a high-molecular-mass precursor protein, due to an impairment in the primary 'break' that normally occurs at the 2A-2B junction. Having all these data present, the study clarified that PDTC disturbs polyprotein processing and replication of two groups of picornaviruses, enteroviruses and cardioviruses, but the underlying mechanism is different.
To conclude, the enzymes that are involved in the picornavirus replication have obviously significant roles in promoting proteolytic activity. Proteolytic processing of viral proteins, have fundamental differences between certain viruses. Non-structural proteins give P1 when cleavages occur, having 3C to play the role of the one that keeps the process going further on, through cis and trans proteolytic cleavages that lie in the inner part of the cell. Protein/polyprotein seems to contribute in virus reproduction. As previously seen, both 2A or 3C are lacking the ability to cleave all the available sites, thus it is an issue that needs to be thoroughly studied. 2A and L proteinases have well-defined positions in the viral polyprotein, flanking the capsid precursor, but they are structurally and biochemically unrelated. Nowadays non-structural proteins of all kind of picornaviruses (2A, 3D, 3C, L) have binary or m multiple purposes. 2A and 3C inhibit the crucial host functions (RNA transcription/protein synthesis) except contributing in virus replication. These enzymes clearly have largely important roles in facilitating proteolytic activity during picornaviral replication and recently it has become more evident that in the proteolytic processing of viral proteins, there are vital differences between certain viruses.
Therapies which combine antiviral drugs with different modes of action and protein targets (e.g. protease and polymerase inhibitors), to reduce the chances of the emergence of drug-resistant mutants, will clarify whether such therapies may work for other viral infections and it will preserve the need to continue to define and characterize additional virus-encoded proteins as targets for antiviral therapy.