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Picornaviruses are small RNA viruses belonging to the family Picornaviridae. They are positive sense single-stranded non-enveloped RNA viruses with an icosahedral capsid causing important diseases in human and animals (Lin et al, 2009). These diseases include the common cold, meningitis, myocarditis, hepatitis, foot-and-mouth-disease, gastroenteritis and poliomyelitis. The Picornavirus is divided into genera including the Enterovirus, Rhinovirus, Hepatovirus, Cardiovirus, Aphthovirus, Erbovirus, etc. which have a simple genomic structure and organisation. The virus particle, made up of capsid proteins, is about 30nm in diameter enclosed within it a 7-9kilobase RNA genome in length. The viral capsid gives the Picornavirus its characteristic shape and size serving multiple functions including (1) protecting the viral RNA from degradation by environmental RNAse, (2) determining host and tissue tropism by recognition of cell-specific cell-membrane receptors, (3) penetrating target cells and delivering the viral RNA into the cell cytoplasm, and (4) selecting and packaging viral RNA (Melnick, 1992). The RNA genome, which encodes a single polypeptide, has the same features as a mammalian mRNA; the 5' untranslated region (UTR), an open reading frame, the 3' untranslated region and a poly (A) tail. Unlike the mammalian mRNA, the viral RNA does not have a capped 5' end but a covalently bound viral protein called VPg. The 5' UTR of the Picornavirus is about 600-1200 nucleotides long. It has a clover-leaf secondary and/or tertiary structure which is important for translation and binds to ribosomes using the internal ribosome entry site (IRES). The internal ribosome entry site (IRES) is aÂ cis-acting RNA sequence that mediates internal entry of the 40S ribosomal subunit on some eukaryotic and viral mRNAs (Buenz and Howe, 2006). Whereas the 3'UTR is ~40-150 nucleotides long, and is important for negative sense cRNA synthesis. The open reading frame of the genome encodes the structural and the non-structural proteins. The single encoded polypeptide is cleaved by viral proteases to smaller viral proteins which is vital for virus replication and infection of the host cell. The polyprotein is initially cleaved into three peptides; P1, P2, and P3. P1 forms the capsid/structural proteins VP0 (VP2 and VP4), VP1 and VP3; P2 forms 2A, 2B and 2C; and P3 forms 3A, 3B, 3C and 3D. The VP1, VP2, VP3 and VP4 forms the capsid proteins. They are well conserved and form the icosahedral structure of the virus (Figure 1). Flanking the capsid proteins in the polyprotein molecule are the L and 2A proteases. They are structurally and biochemically different and share no common ancestry. They more often than not counteract host cell defences (Agol and Gmyl, 2010). The L protease is found in only some Picornavirus. The 2B and 3A proteins are significant for sustainability but less involved in viral replication and between them an ATPase protein, 2C. The 3B protein or VPg encodes the primer used for the negative sense cRNA synthesis and 3D comprises the RNA dependent RNA polymerase which synthesises a new viral RNA stand. Finally, between the 3B and 3D protein is the well conserved major protease of the Picornavirus family, 3C. Markedly, the cleavage into smaller individual viral proteins is not immediately attained. Instead, stable precursors such as 3CD exist (Buenz and Howe, 2006). Amongst the Picornaviridae family, these viral proteins are well conserved with 2A and L proteases, 5' and 3' UTR having significant differences.
For the virus to infect a cell, a number of events must be coordinated and complete. These events include the replication of plus-strand RNA via minus-strand intermediates, RNA translation, proteolytic processing/ cleavage, inhibition of host cell transcription/ translation machinery, virion assembly, and cell lysis (Porter, 1993). From the above statement, proteolytic processing via viral proteases are vital for viral replication, maturation, suppression of host proteins and alteration of host cell structure. Their study is crucial for the understanding of the mechanism used by these viruses to replicate and translate their genomes in a host cell. These important proteases in Picornavirus replication (3C, 2A and L) are discussed in more detail below.
The 3C protease is the major protease in all Picornavirus involved in the processing of the polyprotein and sometimes acts as its precursor 3CD (Tong, 2002). It is a chymotrypsin-like cysteine protease (Seipelt et al, 1999) with the catalytic triad of Histidine (H), Cysteine (C), Glutamic acid (E) or Aspartic acid (D) in its active site. For example, the Human Rhinovirus (HRV) has the H, C, and E catalytic triad while the Hepatitis A virus (HAV) has the H, C, D catalytic triad (Allaire et al, 1994). The 3C proteases have the ability to self-cleave themselves from their precursors on translation of the polyprotein. The 3C protease catalyses the cleavage of the polyprotein in trans as both the N-terminus and C-terminus are far away from the protease active site (Tong, 2002). The 3C protease is encoded by the P3 region of the viral RNA genome, which is also a part of a larger precursor 3CD (the other, 3D, being responsible for the RNA-dependent RNA polymerase) (Figure 1). The 3CD protein is responsible the processing of both the structural and non-structural regions of the polyprotein (P1-P3) but 3C is only involved in the processing of the non-structural regions (P2 and P3) with the exception of Foot-and-Mouth Disease Virus (FMDV), Hepatitis A virus (HAV) and Encephalomyocarditis Virus (EMCV) (Dougherty and Semler, 1993) (Figure 2). In Poliovirus (PV), the 3C protease is highly specific during the cleavage of the polyprotein. It cleaves exclusively at Gln-Gly bonds in the viral substrate. However in other Picornaviruses, 3C is less specific and cleaves at different amino acid pairs like Gln-Ser, Gln-Ala, Gln-Thr, Gln-Ile, Gln-Asn, including Gln-Gly (Lawson and Semler, 1990). In PV infection, the 3C protease has been implicated to alter host cell transcriptional machinery. It was reported to be responsible for the inactivation of RNA polymerase III transcription factor (TFIII) in infected HeLa cells by dephosphorylation and proteolytic cleavage (Clark et al, 1991). The TATA-binding has been reported to be cleaved by 3C protease inhibiting the host cell's RNA polymerase II (Clark et al, 1993), the cyclic AMP-responsive element binding protein (CREB) which leads to loss of the host's DNA binding and transcriptional activity (Yalamanchili et al, 1997a) and the octamer binding transcription factor, Oct-1, leading to the inhibition of its transcriptional activity (Yalamanchili et al, 1997b). The 3C protease is also involved in the inhibition of host cell translation by cleavage of eukaryotic initiation factor 4GI (eIF4GI) in poliovirus (Kuyumcu-Martinez et al, 2004), CV (Chau et al, 2007), FMDV (Strong and Belsham, 2004) and HRV (Aminva et al, 2004). The eIF4GI is a component of the eIF4F complex that mediates the initiation of 5' capped mRNA translation. Another component of the eIF4GI complex is the eIF4GA which is also cleaved by the 3C protease of FMDV (Belsham et al, 2000). The 3C protease also blocks the host's defence systems. In PV infection, the 3C is shown to cleave the p65/RelA subunit of the nuclear factor kappa B (NF-KB), which plays a role in regulating the cell's immune system to infection (Neznanov et al, 2005), Ras-GAP SH3 domain-binding protein (G3BP) which forms stress granules during virus infection (White et al, 2007) and Mitochondrial antiviral signalling protein (MAVS) which activates the host cell innate immune response to viral infection in HAV (Yang et al, 2007). The 3C protease cleaves other cellular and structural proteins. In poliovirus, the 3C protease cleaves the Lupus autoantigen (La) protein of infected cells which may play a role in the stimulation of internal initiation of PV translation in the cytoplasm (Shiroki et al, 1999), the Microtubule-associated protein 4 (MAP-4), a cytoskeletal protein which changes the microtubule system of host cell when infected with PV (Joachims et al, 1995) and Histone proteins, a protein required for structural integrity of the host cell chromatin when infected with FMDV (Tesar and Marquardt, 1990).
The 2A protease is found in some Picornaviruses and it catalyses the release of the release of the structural polyprotein with its cleavage site at the N-terminus of the protein (Toyoda et al, 1986). It is a chymotrypsin-like cysteine protease and the smallest of the family of cysteine proteases (Allaire et al, 1994). The catalytic triad of this protease contains H, C, D in its active site (e.g. HRV), the same as in 3C protease (Petersen et al, 1999). The viral processing of the polyprotein occurs in cis making the protease able to catalyse a cleavage at its N-terminus (Toyoda et al, 1986). In Poliovirus,Â Coxsackie A virus (CV) and Human Rhinovirus (HRV) (Sommergruber et al, 1989), the 2A protease cleaves the polyprotein at its N-terminus at the junction of P1- P2 or VP1- 2A (Figure 2). This cleavage is rapid and occurs during the synthesis of the polyprotein which separates the structural precursor (P1) from the non-structural precursors (P2 & P3). In Aphthovirus (FMDV) and Cardiovirus (EMCV & TMEV) however, this is not the case as the 2A protease cleaves at its C-terminal (Figure 2). Like the Picornavirus 3C protease, the 2A proteases of these viruses are less specific in amino acid pair cleavage; however, preferred pairs are required for efficient proteolytic cleavage. In PV, the preferred cleavage is between Glycine-Tyrosine bonds (Dougherty and Semler, 1993). According to Alvery et al, 1991, it is shown that the 2A proteases can cleave both in trans and cis. Like 3C protease, 2A protease in Picornavirus is also required for the inhibition of host cell translation. In Rhinovirus and Enterovirus, the 2A protease cleaves the p220 protein, an important component of the eukaryotic initiation factor 4F (eIF4F) which interacts with capped cellular mRNA translation (Lloyds et al 1988). This results in the shutoff of host translation machinery. It is also suggested that the 2A protease does not act alone but with another protease. The 2A protease is also involved in the cleavage of eIF4GI in PV, HRV, CV, and FMDV to inhibit cap-dependent translation initiation and also the cleavage of eIF4GII, a homolog of eIF4GI, which results in the shutoff of host cell protein synthesis and apoptotic cell death in PV (Goldstaub et al, 2000) and HRV infection (Svitkin et al, 1999). The 2A protease is important in blocking the host cell transcriptional machinery. In PV, it cleaves the TATA-binding protein (but does not inhibit the host RNA polymerase II) and likewise the poly (A) binding protein (PABP) in PV and CV infection which is important for host cell translation shutoff (Joachims et al, 1999). The 2A protease is also involved in the impairment and disruption of structural proteins. In CV, the 2A protease cleaves a large cytoskeletal protein called Dystrophin which can lead to muscular dystrophy in humans (Badorff et al, 2000) and another structural protein cytokeratin 8, a filament protein, of which 14 amino acid residues are cleaved from the N-terminus of the protein during infection of CV and HRV (Seipelt et al, 2000). The 2A protease cleaves another type of protein- Nucleoporins - which imports/exports nuclear proteins within a cell during infection with PV and HRV. Cleavage of this protein blocks RNA nuclear trafficking inhibiting host cell replication (Castello et al, 2009). Gemin 3, a host factor which is responsible for the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) is also cleaved by 2A protease during PV infection. A reduction of this protein complex leads to spinal muscular atrophy (Almstead and Sarnow, 2007). The multiple function of the 2A protease during synthesis of the viral polyprotein makes it important for viral replication.
The leader protease is also a protease found in some Picornaviruses situated before the capsid precursor proteins (Figure 1). It is an autocatalytic papain-like cysteine protease which catalyses its release from the polyprotein with its cleavage site at the C-terminus of the protein (Strebel et al, 1986). The L protease like 2A cleaves eIF4GI blocking capped translation of host cell mRNA (Tong, 2002). It has the catalytic triad H, C, D (e.g. FMDV) and catalyses its release from the polyprotein by an intermolecular (cis) reaction via its c-terminus (Glaser et al, 2001). In Aphthovirus, the L protease cleaves itself from the polyprotein and also the host cell proteins (Figure 2). In FMDV infection, the L protease is involved in the shutoff of host cell translation. Like the 2A protease of Enterovirus and rhinovirus, the L protease of FMDV is required for the cleavage of p220 subunit of eIF4F complex (Devaney et al, 1988). The L protease is also present in the Cardiovirus genera (EMC and TMEV) but does not contain any recognisable proteolytic motifs or show any cleavage of p220 (Dvorak et al, 2001). The L protease inhibits host cell transcription in a number of ways. In Cardiovirus, the L protease suppresses alpha/beta interferon (IFN-Î±/Î²) expression by inhibiting dimerization of the interferon regulatory factor 3 (IRF-3) (Hato et al, 2007). The L protease of Cardiovirus also inhibits nuclear trafficking. Unlike the 2A protease of PV and HRV which inhibits nuclear protein transport by proteolysis (Castello et al, 2009), the L protease of EMCV inhibits the protein by cytosol-dependent phosphorylation (Porter and Palmenberg, 2009).
Various reports show that Picornavirus non-structural proteases are important in viral replication and infection. They are not only important in proteolysis of the polyprotein but also inhibit essential host cell functions like protein synthesis, RNA transcription and nuclear transport making them multifunctional. The dependence on certain cellular membranes and complexity of Picornavirus replication makes it even more difficult to understand the multifunctionality of these viral non-structural proteases (Lin et al, 2009). The Picornavirus 3C protease can enter the nucleus and function through the precursor 3CD. The 3C protease cleaves a number of transcriptional factors and cellular proteins associated with the host cell mechanism such as the RNA polymerase II and III transcription factor, TATA-binding protein (TBP), Cyclic AMP-responsive element binding protein (CREB), Octamer-binding transcriptional factor (Oct-1), p65/RelA subunit of NF-KB, Ras-GAP SH3 domain-binding protein (G3BP), Mitochondrial antiviral signalling protein (MAVS), Lupus autoantigen (La) proteins, Microtubule-associated protein 4 (MAP-4) and Histone H3 proteins. The 2A protease also inhibits cellular and structural proteins such as Nucleoporins, Small nuclear ribonucleoproteins, Dystrophin and Cytokeratin 8. The L protease, although present in only Aphthovirus and Cardiovirus infected cells, inhibits transcriptional factors and cellular proteins like the inhibiting interferon regulatory factor 3 (IRF-3) and Nucleoporins. However, these proteases also together in inhibiting the host cell translational mechanism which leads to shutoff of mRNA translation. 3C, 2A and L in Picornavirus cleave the eukaryotic initiation factor 4GI (eIF4GI) while 2A and L protease cleave the p220 subunit of eIF4F of some Picornaviruses. 3C and 2A cleave the eIF4GA and poly (A) tail binding protein (PABP) respectively. This goes to show that the hijacking and shutdown of host cell translational mechanism for the replication and hence survival of the Picornavirus is the direct role and function of the viral proteases.