Rebecca Wattam 6-21-2005 0.83 wattam@vbi.vt.edu Arg-Gly-Asp motif of VP1 Extracellular Ligand binding or carrier In 1984 Pierschbacher and Ruoslahti, studying the binding of fibronectin to cells, reported that the tripeptide sequence Arg-Gly-Asp (RGD) was a cellular recognition site on the molecule and that this sequence was also found in the FMDV VP1 protein. The fibronectin receptor was subsequently shown to be part of a large family of transmembrane glycoproteins called integrins. These molecules are type l membrane glycoproteins, consisting of two subunits ({alpha} and β) which are noncovalently bound at the cell surface. They are involved in cell adhesion, cell migration, thrombosis, and lymphocyte interactions. In FMDV, while sequences surrounding the RGD sequence within the G-H loop are variable, the RGD sequence itself is highly conserved. The first indication that the RGD sequences might be involved in the virus-receptor interaction came from studies showing that small peptides containing RGD could inhibit the binding of virus to cells. Direct genetic evidence for this interaction was obtained by mutating or deleting the RGD sequence in infectious cDNA clones, resulting in viral particles which were noninfectious, could not adsorb to susceptible cells, and could not cause disease in susceptible animals (Grubman and Baxter, 2004).<p> FMDV initiates infection in cultured cells by binding to any of four members of the {alpha}V subgroup of the integrin family of cellular receptors ({alpha}Vβ1, {alpha}Vβ3, {alpha}Vβ6, and {alpha}Vβ8) via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the βG-βH loop of VP1 (O'Donnell et al., 2005). Cell Surface Receptor Cell membrane Ligand binding or carrier Two classes of cell surface receptors that mediate FMDV infection have been identified. These are the integrins and heparan sulfate proteoglycans (HSPGs). The ability to use HSPGs as receptors appears to be restricted to strains of FMDV that have been multiply passaged through cultured cell lines, and presently there is no convincing evidence of a role for HS in cell entry by field viruses. Instead, field viruses are dependent on integrin receptors to initiate infection in vitro, and integrins are believed to be the receptors used in the infected animal. Recently, two independent studies have shown that certain strains of FMDV can infect cultured cells via an entry pathway that is independent of both integrins and cellular HS, implying the existence of a third, as yet unidentified receptor family (Jackson et al., 2004).<p> FMDV initiates infection in cultured cells by binding to any of four members of the {alpha}V subgroup of the integrin family of cellular receptors ({alpha}Vβ1, {alpha}Vβ3, {alpha}Vβ6, and {alpha}Vβ8) via a highly conserved arginine-glycine-aspartic acid amino acid sequence motif located within the βG-βH loop of VP1. We have recently shown that the {alpha}Vβ6 integrin acts as a high-affinity receptor for the virus while {alpha}Vβ3 interacts with virus with a much lower affinity. In addition, viruses of the type A serotype utilize both the {alpha}Vβ3 and {alpha}Vβ6 integrins as receptors in cultured cells, while serotype O viruses have an affinity for the {alpha}Vβ6 integrin (O'Donnell et al., 2005).<p> It was subsequently shown that among the myriad of integrins, isoforms ?Vβ3 and ?Vβ6 most likely serve as cellular receptors for FMDV. Antibody-bound FMDV can also infect cells via Fc receptor-mediated adsorption or through an engineered artificial receptor consisting of a single-chain anti-FMDV monoclonal antibody fused to ICAM-1 (Mason et al., 2003).<p> It appears that FMDV is promiscuous with respect to receptor utilization and can utilize regions of the virion other than the G-H loop of 1D to bind to (and infect) cells in culture (Mason et al., 2003). FMD virus Bound to Cell Surface Cell membrane Other In cell cultures expressing both the {alpha}Vβ3 and {alpha}Vβ6 integrins, virus adsorbed to the cells at 4 C appears to colocalize almost exclusively with the {alpha}Vβ6 integrin. Upon shifting the infected cells to 37 C, FMDV capsid proteins were detected within 15 min after the temperature shift, in association with the integrin in vesicular structures that were positive for a marker of clathrin-mediated endocytosis (O'Donnell et al., 2005).<p> FMDV binds rapidly to cells in culture at both 4 and 37 C by using a limited number of receptor sites, which has measured at between 10(3) and 10(4) per cell (Grubman and Baxt, 2004). FMD virus in Endosome Phagolysosome Other Following the binding of FMDV to the cell surface, we analyzed the entry route of the virus into the cell. FMDV appears to be internalized, in association with the {alpha}Vβ6 integrin, into a structure resembling an endocytic vesicle and is translocated from the plasma membrane to the perinuclear region within 1 h p.a. (O'Donnell et al., 2005). Uncoated FMD virus in Endosome Phagolysosome Other The virus is thought to enter endosomes, leading to capsid uncoating in the acidic environment of this compartment (Miller et al., 2001). <p> While the penetration and uncoating of FMDV have not been studied in great detail, there have been some observations which suggest possible mechanisms of how they might occur. We and others have shown that after adsorption to the cell surface, the 140S virion breaks down into 12S pentameric subunits, releasing the RNA. This breakdown does not occur at the cell surface, since particles which are eluted from the cell after adsorption are fully infectious and still sediment at 140S. By using a series of lysosomotropic agents, which raise the pH of intracellular endosomes, it has been demonstrated that the virus probably breaks down upon entering an acidic endosome (Grubman and Baxt, 2004).<p> Breakdown of the virion in the acidic endosome may be necessary, but is not sufficient, for infection since RNA containing non-infectious provirion structures with uncleaved 1AB are as acid sensitive as mature virions (Mason et al., 2003).<p> Unlike other picornaviruses, FMDV is susceptible to low pH-induced (pH below 6.5) disassembly. It has been suggested that a His-rich region at the 1B/1C quasi 2-fold interface is responsible for the acid-induced disassembly, since protonation of these residues at this pH could produce repulsive forces opening the capsid (Mason et al., 2003). Genomic RNA in Cytoplasm Cytoplasm Other Following virus attachment and entry into the cell, the RNA genome is delivered into the cytoplasm and translation of the genome is required to produce the viral proteins that are necessary for virus assembly and RNA replication. The genome encodes a single large polyprotein that is processed, largely by internal trans-acting proteases, to produce about 12 mature proteins plus various precursors (some of these have distinct functions). The proteins encoded within the P1 region form the capsid, while proteins encoded in the P2 and P3 regions are required for RNA replication (Nayak et al., 2005).<p>FMDV genomic RNA is about 8.3 kb in length. All of the viral RNA is of the same length; no sub-genomic mRNAs are produced by picornaviruses (Belsham, 2005).<p>The 5' end of the genome RNA is covalently linked to VPg, and the 3' end has a genetically coded poly(A) tail. Thus, the viral RNA-dependent RNA polymerase (3Dpol) must distinguish between viral RNAs and cellular mRNAs, which also contain 3'-terminal poly(A) tracts. In addition, since the mRNA and the genome RNA are the same molecule, with the exception of the genome-linked VPg, there must be a mechanism to distinguish RNAs which are bound for the ribosome and those which will be packaged into virion particles (Grubman and Baxt, 2004). Genomic RNA minus VpG Cytoplasm Other The genome-linked protein VPg is cleaved by a cellular enzyme prior to translation of the incoming RNA; however, protein synthesis initiation complexes can be formed with mRNA containing VPg. (Grubman and Baxt, 2004). VPg (3B) Other The genome-linked protein VPg is cleaved by a cellular enzyme prior to translation of the incoming RNA (Grubman and Baxt, 2004).<p>There is less information concerning the properties of the VPg on foot-and-mouth disease virus (FMDV) RNA. It is known that this protein is small (molecular weight 5,000 by SDS-gel electrophoresis) and, unlike poliovirus VPg, contains methionine. The protein is not needed for the infectivity of the RNA nor for the efficient translation of the RNA in vitro (King et al., 1980). VPg (3B) Other VPg uridylylation is required for replication initiation, and uridylylated VPg (VPg-pU-pU) is utilized as a primer for the initiation of RNA synthesis at the 3' poly(A) tract of the viral RNA. The VPg protein is covalently linked to the 5' end of both positive- and negative-strand picornavirus RNAs, suggesting that VPg has a functional role in RNA replication. VPg uridylylation is carried out by the enzymatic activity of the 3D polymerase, which covalently couples uridine nucleotides to a conserved tyrosine residue in the VPg protein. Although the 3D RNA polymerase is responsible for uridylylating the VPg primer and elongating RNA strands from the 3' poly(A) template in vitro, the process may involve other viral or host proteins for RNA replication initiation in vivo (Bedard and Semler, 2004).<p>Each of the picornavirus cre structures contains a conserved mofit of AAACA located within the loop region. This motif acts as the template for the uridylylation of VPg (3B) by the viral polymerase to produce VPgpU and/or VPgpUpU. These products act as the primers for the initiation of viral RNA synthesis, thus explaining the presence of VPG at the 5' terminus of both positive and negative sense RNA transcripts (Belsham, 2005). Polyprotein Other The RNA codes for only one long open reading frame. Translation proceeds primarily from a single strong initiation site and produces a giant precursor polyproteins (M(r) approximately 250,000) that is divided into three regions: P1, P2, and P3. The polyprotein is processed in a series of proteolytic cleavage steps to yield mature virion capsid proteins as well as other noncapsid viral proteins (Arnold et al., 1987).<p>The single large open reading frame encodes a polyprotein which is never observed. It is processed by virus-encoded proteases during synthesis. Many different precursors can be generated during this processing of the polyprotein (Belsham, 2005). Leader (L) protein Other Enzyme The first component of the FMDV polyprotein is the Leader (L) protein. FMDV is unique in having a protease as the Leader protein. The L protein is a papain-like cysteine protease and it has at least two distinct activities. It cleaves itself from the rest of the viral polyprotein at the L/P1 junction, and it also includes the cleavage of the translation initiation factor eIF4G; this results in the inhibition of cap-dependent protein synthesis (Belsham, 2005). <p> A papain-like proteinase, it mediates autocatalytic cleavage of itself from P1 and cleaves the host protein eIF4GI, resulting in the shut-off of host cap-dependent mRNA translation (Fry et al., 2005).<p>The Lpro protein is coded for by the 5'-end of the ORF. Within the Lpro-coding region of all seven serotypes of FMDV are two in-frame AUG codons that encode proteins termed Lab and Lb. Although both products have been detected in vitro as well as in infected cells, studies by Cao et al. suggest that the Lb protein (synthesized from the second AUG) is the major protein synthesized in vivo, since viable viruses can be recovered from synthetic genomes containing mutations in the first AUG, but not the second (Mason et al., 2003).<p>Currently it may be considered that the FMDV IRES directs ribosome attachment to the viral RNA either just upstream or just downstream of the Lab initiation site. Some ribosomes land upstream of the Lab site and can then initiate protein synthesis at this point, but some may fail to do so and then scan along the RNA until the Lb site is reached. The ribosomes which land downstream of the Lab site presumably just migrate along the RNA to initiate translation at the Lb site (Belsham, 2005).<p>The Lpro-deleted virus should be less cytopathic than WT virus, since Lpro cleavage of eIF4G results in shut-off of host protein synthesis. However, preliminary characterization of the leaderless virus in BHK cells demonstrated that A12-LLV2 replicated at only a slightly slower rate than WT virus, and resulted in approximately 10-fold lower yields with a slightly smaller plaque size than WT virus. These same studies failed to show a marked difference in the pathogenesis of WT and leaderless virus in suckling mice. Interestingly, the serotype A12 leaderless virus displayed a 100 000-fold lower ability than the WT virus to cause lesions when injected intradermally in the tongue of cattle, and failed to cause disease when inoculated by this route. In addition, cattle or swine inoculated subcutaneously with high doses of leaderless virus and co-housed with uninoculated control animals, displayed no clinical signs of FMD, and there was no evidence of transmission to the uninoculated co-housed animals. In contrast, cattle and swine inoculated with WT A12 virus developed typical signs of FMD and the virus was transmitted to co-housed uninoculated animals. These results indicate that the virulence of FMDV is correlated with the presence of Lpro, and that Lpro is an essential gene for causing disease and permitting transmission in livestock hosts (Mason et al., 2003).<p>Further evidence for a critical role for Lpro in pathogenesis came from studies in which cattle were exposed to aerosol containing high doses of WT or leaderless virus. Animals exposed to WT virus developed clinical FMD (fever and vesicles) by 72 h postexposure (hpe), while leaderless virus-exposed animals never developed detectable disease. By 24 hpe the WT virus-exposed animals had histologically altered respiratory bronchioles and virus-specific in situ hybridization (ISH) signals in the bronchioles, and by 72 hpe these animals had positive ISH signals in epidermal sites that corresponded to visible lesion development. In contrast, the leaderless virus-exposed animals had no discernable histological changes in the lung, significantly fewer ISH signals around respiratory bronchioles at both 24 and 72 hpe, and no detectable lesions or ISH signals in epithelial tissue. Thus, in contrast to WT virus infection, leaderless virus remained localized to limited areas of the lung and did not spread to sites of virus predilection. Interestingly, when a leaderless virus was constructed from a highly virulent FMDV with a serotype O1 capsid, the virus produced mild disease in pigs, indicating that leaderless viruses could not be utilized as genetically engineered live attenuated vaccines (Mason et al., 2003).<p>The L protease is not essential for FMDV replication; a mutant of the virus lacking the entire Lb coding sequence has been made. The mutant virus replicates in BHK cells (albeit more slowly than the parental virus) but is attenuated in cattle. The failure to rapidly shut off host cell protein synthesis may permit cells to mount a more efficient anti-viral response (Belsham, 2005). Lpro Other Enzyme Lpro cleaves itself from the growing polypeptide chain at its carboxyl terminus and degrades host translation initiation factor eIF4G. Sequence alignment studies suggested that Lpro is a papain-like proteinase, and additional studies utilizing proteinase inhibitors, genetic engineering, and X-ray crystallography have substantiated this original suggestion. Moreover, the crystallographic studies, which identified crystal forms of dimers of Lpro, suggested that Lpro acts as a trans-proteinase when it releases itself from the viral polyprotein (Mason et al., 2003). P1-2A Precursor Other The P1-2A precursor is processed by the 3C protease to yield VP0 (1AB), VP3 (1C) and VP1 (1D). These are the components of natural empty capsids, and 60 copies of each protein will self-assemble to form particles (Belsham, 2005).<p>The structural proteins are encoded by the P1 region of the genome, and processing of the P1/2A precursor is accomplished by 3Cpro leading to the formation of protomers containing one copy each of 1AB, 1C, and 1D (Mason et al., 2003). P1 Other The P1/2A cleavage to form the structural protein precursor, P1, is performed by the 3C protease (Fry et al., 2005).<p>The structural proteins are encoded by the P1 region of the genome, and processing of the P1/2A precursor is accomplished by 3Cpro leading to the formation of protomers containing one copy each of 1AB, 1C, and 1D (Mason et al., 2003). VP1 (1D) Other Other The P1-2A precursor is processed by the 3C protease to yield VP0 (1AB), VP3 (1C) and VP1 (1D). These are components of the natural empty capsids (Belsham, 2005).<p>The 1D region in the viral RNA codes for a major antigenic site consisting of the GH loop (140 to 160 amino acids) and a minor antigenic site (205 to 210 amino acids) at the C terminus of VP1 protein (Nagendrakumar et al., 2005).<p>In all FMDVs the C-terminus and the GH loop of VP1 are highly exposed regions which are central to both antigenicity and receptor binding. In type O FMDV, for example, trypsin cleavage of VP1 within the GH loop and at the C-terminus does not affect the integrity of the virus particle but abolishes the ability of the virus to attach to cells and greatly reduces immunogenicity (Fry et al., 2005). VP1 (1D) Other Other Cytoplasmic structures staining for viral proteins form at perinuclear sites close to a dispersed Golgi apparatus. The viral non-structural protein 2C co-localizes with VP1, 3A and 3D within these structures, but not with proteins associated with organelles of the host cell secretory pathway, including the ER, the ER to Golgi intermediate compartment (ERGIC), Golgi complex, trans-Golgi network (TGN) or lysosomes. The results suggest that these membranes of the secretory pathway are not used by the virus as a platform for replication and assembly, or that the FMDV-induced structures are formed by membrane rearrangements which involve exclusion of organelle-specific protein markers (Knox et al., 2005). VP3 (1C) Other Other The P1-2A precursor is processed by the 3C protease to yield VP0 (1AB), VP3 (1C) and VP! (1D). These are components of the natural empty capsids (Belsham, 2005).<p>The cores of proteins 1B, 1C, and 1D consist of a highly conserved eight-stranded ?-barrel that is a hallmark of the capsid proteins of a large number of icosahedral viruses that affect both animals and plants. As with other picornaviruses, 1A, which appears to hold a structural conformation similar to N-terminal portions on 1C and 1D, is myristylated and buried within the virion. Therefore, the entire capsid surface is covered by portions of the other three proteins (Mason et al., 2003). VP0 (1AB) Other Other The P1-2A precursor is processed by the 3C protease to yield VP0 (1AB), VP3(1C) and VP! (1D). These are components of the natural empty capsids (Belsham, 2005).<p>VP4 exists initially as an N-terminal extension of VP2 in the precursor VP0 (Curry et al., 1997).<p>The final step in the transition of the provirion to the mature virion, is the cleavage of the 1AB protein to 1A and 1B, which occurs by unknown mechanisms and is dependent on the presence of the viral RNA. There has been a report of FMDV 1AB cleavage in empty capsids, however, this cleavage occurred at an abnormal site, suggesting that RNA is required for normal provirion maturation. These same studies suggested that the maturation cleavage mechanism involves a conserved His residue in 1B which activates local water molecules leading to a nucleophilic attack on the scissile bond resulting in 1AB cleavage (Mason et al., 2003).<p>VP0 peptides are cleaved into VP2 and VP4, thus completing the final stages of both processing and assembly. The VP0 digestion takes place deep within a maturing particle, inaccessible to exogenous proteases, including proteases 3C and 2A (Arnold et al., 1987). VP0 (1AB) Other Other The final cleavage within picornaviral polyproteins is probably in VP0 (peptide 1AB) to give VP4 (1A) and VP2 (1B) (Arnold et al., 1987).<p>Myristylation of 1AB is necessary for efficient assembly of capsid structures (Mason et al., 2003).<p>The final step in the transition of the provirion to the mature virion, is the cleavage of the 1AB protein to 1A and 1B, which occurs by unknown mechanisms and is dependent on the presence of the viral RNA. There has been a report of FMDV 1AB cleavage in empty capsids, however, this cleavage occurred at an abnormal site, suggesting that RNA is required for normal provirion maturation. These same studies suggested that the maturation cleavage mechanism involves a conserved His residue in 1B which activates local water molecules leading to a nucleophilic attack on the scissile bond resulting in 1AB cleavage (Mason et al., 2003).<p>VP0 peptides are cleaved into VP2 and VP4, thus completing the final stages of both processing and assembly. The VP0 digestion takes place deep within a maturing particle, inaccessible to exogenous proteases, including proteases 3C and 2A (Arnold et al., 1987). VP2 (1B) Other Other The virion is a 140S particle consisting of a single-stranded RNA genome and 60 copies each of four structural proteins (VP1 [1D], VP2 [1B], VP3 [1C], and VP4 [1A]) (Grubman and Baxt, 2004).<p>The three-dimensional arrangements of the structural proteins within the virion provide the antigenic sites that elicit responses to vaccination or infection. In addition, these structures mediate binding to cell receptors, entry of the genome into cells, and determine the stability of the capsid to environmental factors. All of these functions influence how effectively the virus is able to spread within the host and between hosts, and hence are important molecular determinants of virulence. The cores of proteins 1B, 1C, and 1D consist of a highly conserved eight-stranded ?-barrel that is a hallmark of the capsid proteins of a large number of icosahedral viruses that affect both animals and plants. As with other picornaviruses, 1A, which appears to hold a structural conformation similar to N-terminal portions on 1C and 1D, is myristylated and buried within the virion. Therefore, the entire capsid surface is covered by portions of the other three proteins (Mason et al., 2003).<p>Unlike other picornaviruses, FMDV is susceptible to low pH-induced (pH below 6.5) disassembly. It has been suggested that a His-rich region at the 1B/1C quasi 2-fold interface is responsible for the acid-induced disassembly, since protonation of these residues at this pH could produce repulsive forces opening the capsid (Mason et al., 2003).<p>The newly created termini of VP2 and VP4 make interactions that reinforce the connection between pentameric subunits (Curry et al., 2004). VP4 (1A) Other Other The virion is a 140S particle consisting of a single-stranded RNA genome and 60 copies each of four structural proteins (VP1 [1D], VP2 [1B], VP3 [1C], and VP4 [1A]) (Grubman and Baxt, 2004).<p>The three-dimensional arrangements of the structural proteins within the virion provide the antigenic sites that elicit responses to vaccination or infection. In addition, these structures mediate binding to cell receptors, entry of the genome into cells, and determine the stability of the capsid to environmental factors. All of these functions influence how effectively the virus is able to spread within the host and between hosts, and hence are important molecular determinants of virulence. The cores of proteins 1B, 1C, and 1D consist of a highly conserved eight-stranded ?-barrel that is a hallmark of the capsid proteins of a large number of icosahedral viruses that affect both animals and plants. As with other picornaviruses, 1A, which appears to hold a structural conformation similar to N-terminal portions on 1C and 1D, is myristylated and buried within the virion. Therefore, the entire capsid surface is covered by portions of the other three proteins (Mason et al., 2003).<p>The whole of VP4 is associated with the interior face of the capsid. VP4 exists initially as an N-terminal extension of VP2 in the precursor VP0 (Curry et al., 1997). VP2 (1B) and VP4 (1A) Other Other The virion is a 140S particle consisting of a single-stranded RNA genome and 60 copies each of four structural proteins (VP1 [1D], VP2 [1B], VP3 [1C], and VP4 [1A]) (Grubman and Baxt, 2004).<p>The three-dimensional arrangements of the structural proteins within the virion provide the antigenic sites that elicit responses to vaccination or infection. In addition, these structures mediate binding to cell receptors, entry of the genome into cells, and determine the stability of the capsid to environmental factors. All of these functions influence how effectively the virus is able to spread within the host and between hosts, and hence are important molecular determinants of virulence. The cores of proteins 1B, 1C, and 1D consist of a highly conserved eight-stranded ?-barrel that is a hallmark of the capsid proteins of a large number of icosahedral viruses that affect both animals and plants. As with other picornaviruses, 1A, which appears to hold a structural conformation similar to N-terminal portions on 1C and 1D, is myristylated and buried within the virion. Therefore, the entire capsid surface is covered by portions of the other three proteins (Mason et al., 2003).<p>Unlike other picornaviruses, FMDV is susceptible to low pH-induced (pH below 6.5) disassembly. It has been suggested that a His-rich region at the 1B/1C quasi 2-fold interface is responsible for the acid-induced disassembly, since protonation of these residues at this pH could produce repulsive forces opening the capsid (Mason et al., 2003)<p>The newly created termini of VP2 and VP4 make interactions that reinforce the connection between pentameric subunits (Curry et al., 2004).<p>The whole of VP4 is associated with the interior face of the capsid. VP4 exists initially as an N-terminal extension of VP2 in the precursor VP0 (Curry et al., 1997). 2A Other Enzyme 2A, (an 18-amino-acid peptide), autocatalytically removes itself from the P2 polyprotein, and remains associated with the P1 precursor. There has been a suggestion that this cleavage is not a proteolytic event but rather is a modification of the translational machinery by the 2A peptide which allows the release of P1-2A from the ribosome while permitting the synthesis of the downstream proteins to proceed. This hypothesis, however, has not been confirmed by other laboratories. Nevertheless, the 2A peptide has been used in nonviral systems to cleave foreign genes from polyproteins (Grubman and Baxt, 2004).<p>It has been shown that both the FMDV 2A peptide and the 19 C-terminal amino acids of the cardioviral 2A protein, along with the first amino acid of the 2B protein can mediate cleavage in artificial polyprotein systems. Interestingly, however, this FMDV 2A-mediated artificial polyprotein cleavage did not occur in a prokaryotic system. Further studies of this phenomenon indicated that in this artificial system, the proteins synthesized upstream of the 2A sequence were always present in greater molar excess than the proteins downstream of the 2A sequence. This finding led to the hypothesis that the 2A 2B cleavage event is not a proteolytic event, but rather a modification of the translational machinery by the 2A peptide which allows the release of the protein-2A from the ribosome while permitting the synthesis of the downstream proteins to proceed. While this hypothesis explains some of the anomalies of the 2A cleavage reaction, it awaits further confirmation. Nevertheless, the fact that such a short peptide has an apparent proteolytic function has been utilized in diverse systems to produce foreign genes from polyproteins in a variety of host cells (Mason et al., 2003).<p>The small 2A peptide, during its translation, interacts with the exit tunnel of the ribosome to induce the "skipping" of the last peptide bond at the C-terminus of 2A. The crucial point is that the ribosome is able to continue translating the downstream gene, after releasing the first protein fused in its C-terminus to 2A. This type of sequence has been termed CHYSEL (cis-acting hydrolase element) (de Felipe, 2004). 2A Other Enzyme 2A, (an 18-amino-acid peptide), autocatalytically removes itself from the P2 polyprotein, and remains associated with the P1 precursor. There has been a suggestion that this cleavage is not a proteolytic event but rather is a modification of the translational machinery by the 2A peptide which allows the release of P1-2A from the ribosome while permitting the synthesis of the downstream proteins to proceed. This hypothesis, however, has not been confirmed by other laboratories. Nevertheless, the 2A peptide has been used in nonviral systems to cleave foreign genes from polyproteins (Grubman and Baxt, 2004). P2 Precursor (P2BC) Other The FMDV 2BC precursor is processed to 2B and 2C by the 3C protease. Little is known about the function of these proteins (Belsham, 2005).<p>The P2 region contains two nonstructural proteins of unknown function, 2B and 2C (Vakharia et al., 1987). 2B Other Other The picornaviral 2B and 2C proteins have been implicated in virus-induced cytopathic effects. Protein 2B has been shown to enhance membrane permeability and block protein secretory pathways, and both 2B and 2C localize to ER-derived outer surface vesicles which are sites of genome replication (Mason et al., 2003).<p>Although the functions of the FMDV 2B and 2C proteins are unknown, preliminary work suggests that, similar to those of other picornaviruses, they localize to endoplasmic reticulum (ER)-derived vesicles, the sites of viral genome replication (Carrillo et al., 2005).<p>The 2B protein has been shown to enhance membrane permeability and block protein secretion, but its role in RNA synthesis is not clear (Grubman and Baxt, 2004). 2C Other Other The picornaviral 2B and 2C proteins have been implicated in virus-induced cytopathic effects. Protein 2B has been shown to enhance membrane permeability and block protein secretory pathways, and both 2B and 2C localize to ER-derived outer surface vesicles which are sites of genome replication (Mason et al., 2003).<p>In FMDV-infected cells, protein 2C was detected in aggregates located at the cells periphery by immunoflourescence, consistent with a co-localization with membrane-bound replication complexes, and Lubroth and Brown have reported that 2C is absent from clarified virus stocks used for vaccine preparation, which is also consistent with membrane association (Mason et al., 2003).<p>Direct evidence for the role of the FMDV 2C protein in replication has come from studies using the antiviral compound, guanidine hydrochloride. This compound has been shown to inhibit viral RNA synthesis in picornavirus-infected cells, work with FMDV was the first to show a physical change (altered isoelectric point) in the 2C protein of a guanidine-resistant (gr) mutant. The importance of the 2C mutations in the gr phenotype was confirmed by analyzing the genetic and biochemical properties of a panel of recombinant viruses generated using classical methodology. For poliovirus, the 2C protein was also identified as the site of guanidine action, and Barton and Flanegan (1997) have shown that poliovirus 2C is required for initiation of negative-strand RNA synthesis. The 2C proteins of all picornaviruses contain conserved nucleoside triphosphate-binding and helicase motifs, although no helicase activity has been demonstrated for any 2C protein. The protein has been demonstrated to have ATPase and GTPase activity and the ATPase activity has been shown to be inhibited by guanidine. Since the 2C proteins of picornaviruses are highly conserved, it is assumed that FMDV 2C should possess most of these activities (Mason et al., 2003). 2C-hypothetical link Other Other For plus-strand synthesis to proceed, the RF must be unwound. The mechanism for this is also unclear. The picornavirus 2C protein both has ATPase activity and contains helicase motifs, but helicase activity has not been demonstrated. It has been shown that 2C and a cellular protein (p38) bind to the minus-strand 3' stem-loop, and this may act to destabilize the RF molecule (Grubman and Baxt, 2004). P3 Precursor Other Other The P3 region includes four nonstructural proteins: 3A, of unknown function; 3B (3B1, 3B2, and 3B3), the three genome-linked VPg proteins; 3C, a protease; and 3D, the RNA polymerase (Vakharia et al., 1987). P3 Intermediates Other 3ABC: FMDV-3ABC is rapidly degraded after synthesis and associates to a detergent-insoluble fraction in FMDV infected cells (Capozzo et al., 2002).<p>Cells were either pulse-labeled for 7 min with 35S methionine or pulsed and then incubated for 1 h in the presence of a 100-fold excess of methionine. Precursor P3 and 3ABC are the major species detected immediately after the pulse while 3CD and protease 3C are the predominant polypeptides immunoprecipitated after the 1 h chase. 3ABC is an early post-translational product of P3 and a likely intermediate in the production of protease 3C in FMDV infected cells. The proteolytic potential and the particular subcellular location of 3ABC led us to suggest that it could be involved in the cleavage of the highly modified amino terminus of histone H3, and the inhibition of host RNA transcription during FMDV infections. Recombinant FMDV-3ABC expressed in bacteria resulted however in a stable polypeptide and neither cys- nor trans-protease activity has been associated with this protein. This is inconsistent with the proposed proteolytic activity of 3ABC suggesting that it could be a by-product of P3 that results from non-specific degradation during FMDV infection (Capozzo et al., 2002).<p>3AB, 3ABB, 3ABBB: 3A forms part of the stable intermediates that include 3AB and 3ABB (Mason et al., 2003).<p>Direct labeling and detection of VPg polypeptides are extremely difficult because of their small size, the lack of sulfur-containing amino acids, their basic hydrophilic character that results in their leaking from polyacrylamide gels during fluorography or fixation, and the rapid degradation of free VPg polypeptides in the infected cell. Therefore, we used an indirect method to study the proteolytic processing of the FMDV VPg precursor proteins by analyzing VPg fusions with the labeled neighboring polypeptide 3A. VPg is relatively stably linked to this membrane-anchoring protein, forming precursor polypeptides 3AB(1), 3AB(12), and 3AB(123) (Falk et al., 1992).<p>Analysis of all immunoprecipitations of VPg polypeptides, synthesized in vitro or in vivo, revealed a double-band pattern for all three VPgs (products 3AB(1), 3AB(12), and 3AB(123) (Falk et al., 1992).<p>3CD: In enterovirus- and rhinovirus-infected cells 3CDpro is a major intermediate, and has specific altered cleavage activities relative to the 'mature' 3Cpro. In addition, 3CD is known to participate in formation of ribonucleoprotein complexes, and 3CD binding to RNA is thought to influence both replication and translation. In FMDV-infected cells, 3CD is found at variable levels, depending on the virus subtype. However, in FMDV type A12-infected cells, 3CD is processed relatively rapidly, hence some of the activities mediated by 3CDpro for other picornaviruses may be carried out by 3Cpro or 3Dpol of FMDV (Mason et al., 2003). 3A Other Other The P3 domain also produces protein 3B, a primer for picornaviral RNA synthesis and protein 3A, a membrane bound protein in polio virus (PV) and hepatitis A virus (HAV) infected cells and linked to the attenuation of virulence and membrane formation in foot and mouth disease virus (Capozzo et al., 2002).<p>FMDV-3A is 139 amino acids-long and has a relatively small hydrophobic tract, 12% of the sequence, located to the center of the polypeptide. Both PV (polio virus) and HAV (hepatitis A virus) 3A proteins are smaller polypeptides, 87 and 75 amino acids, respectively, with hydrophobic sequences located at the C-terminus and representing up to 25% of the total protein (Capozzo et al., 2002).<p>The 3A protein has hydrophobic sequences which are believed to anchor it to membranes, and this may be the means by which RNA replication is localized to membrane vesicles. The 3A protein may also serve to deliver the 3B peptides to the sites of RNA replication. Certain strains of FMDV have been isolated that contain in-frame deletions within the 3A coding sequence, and these strains are attenuated in cattle but remain highly pathogenic in pigs. This presumably reflects some differences between the two species in the interaction of 3A with cellular factors (Belsham, 2005). 3B1 (VPg1) Other Other FMDV uniquely expresses 3 distinct copies of the viral protein 3B (3B1, 3B2, and 3B3, or VPg1-3) as the primer for RNA replication, this feature is conserved in all strains of FMDV. Each of these different peptides has been shown to be used during virus replication and to be linked to genomic RNA. Deletion of the individual copies of VPg has a deleterious effect on RNA replication. In particular, deletion of the 3B3 coding sequence resulted in a nonviable virus, but this defect was attributed to impaired proteolytic processing of the polyprotein (Nayak et al., 2005). 3B2 (VPg2) Other Other The role of VPg in FMDV seems to be more complex than its role in the other picornaviruses. Sequence analysis of the FMDV genome revealed three VPg genes arranged in tandem and designated VPg1, VPg2, and VPg3, an astonishing redundancy considering the small genome size. All other picornavirus groups have only one VPg-coding region. The three VPg genes are related but distinctly different from one another and were found in an equimolar ratio in virions, suggesting equivalent functions in vivo. Since no specialized function of each individual VPg was found, a selective advantage for a high VPg gene copy number in FMDV was presumed (Falk et al., 1992). 3B3 (VPg3) Other Other FMDV uniquely expresses 3 distinct copies of the viral protein 3B (3B1, 3B2, and 3B3, or VPg1-3) as the primer for RNA replication, this feature is conserved in all strains of FMDV. Each of these different peptides has been shown to be used during virus replication and to be linked to genomic RNA. Deletion of the individual copies of VPg has a deleterious effect on RNA replication. In particular, deletion of the 3B3 coding sequence resulted in a nonviable virus, but this defect was attributed to impaired proteolytic processing of the polyprotein (Nayak et al., 2005).<p>Priming of RNA synthesis requires 3B (VPg) (Belsham, 2005). 3C Other Enzyme As demonstrated for other picornaviruses the 3Cpro protein of FMDV was identified as a proteinase and is responsible for most cleavages in the viral polyprotein. Other than autocatalytic cleavage of Lpro from P1, the 2A cleavage between P1 and P2, and the maturation cleavage of 1AB to 1A and 1B, all other cleavages are performed by 3Cpro or a 3Cpro-containing precursor. However, unlike protein processing for poliovirus, in which 3CDpro has been implicated as the major viral proteinase in structural protein cleavage, FMDV 3Cpro can efficiently process all ten cleavage sites in the FMDV polyprotein. Moreover, unlike poliovirus, in which 3Cpro cleavage occurs exclusively between Gln-Gly sites, the FMDV 3Cpro cleavage sites show great heterogeneity, with cleavage occurring between multiple dipeptides, including Gln-Gly, Glu-Gly, Gln-Leu, and Glu-Ser (Mason et al., 2003).<p>Recently it has been demonstrated that FMDV 3Cpro can also cleave eIF4A, which is part of the cap-binding complex and functions as an RNA helicase. It was also shown that 3Cpro can cleave eIF4G late in the infectious cycle, albeit at different sites than Lpro-mediated cleavage. These cleavages occur later in the infection cycle than the Lpro-mediated cleavage of eIF4G and it was speculated that these events may be unfavorable for translation of viral RNA (Mason et al., 2003). 3C Enzyme Once 3C is self-cleaved from the P3 precursor protein, it is responsible for key processing events to generate replication proteins within the P2 and P3 precursor proteins (Bedard and Semler, 2004). 3C Enzyme Once 3C is self-cleaved from the P3 precursor protein, it is responsible for key processing events to generate replication proteins within the P2 and P3 precursor proteins (Bedard and Semler, 2004).<p>The proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C (Vakharia et al., 1987). 3C Enzyme Once 3C is self-cleaved from the P3 precursor protein, it is responsible for key processing events to generate replication proteins within the P2 and P3 precursor proteins (Bedard and Semler, 2004).<p>The FMDV 2BC precursor is processed to 2B and 2C by the 3C protease (Belsham, 2005). 3C Enzyme Once 3C is self-cleaved from the P3 precursor protein, it is responsible for key processing events to generate replication proteins within the P2 and P3 precursor proteins (Bedard and Semler, 2004).<p> The FMDV P3 precursor is separated from P2 and processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (VPg), 3C protease and 3D polymerase plus a variety of intermediates (e.g. 3CD) (Belsham, 2005). 3C Enzyme Once 3C is self-cleaved from the P3 precursor protein, it is responsible for key processing events to generate replication proteins within the P2 and P3 precursor proteins (Bedard and Semler, 2004).<p> The FMDV P3 precursor is separated from P2 and processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (VPg), 3C protease and 3D polymerase plus a variety of intermediates (e.g. 3CD) (Belsham, 2005). 3C Enzyme Protein 2C has been found in membranous aggregates along the periphery of FMDV-infected cells and has been directly implicated in FMDV RNA synthesis by using the picornavirus RNA synthesis inhibitor guanidine hydrochloride (Grubman and Baxt, 2004).<p> Cytoplasmic structures staining for viral proteins form at perinuclear sites close to a dispersed Golgi apparatus. The viral non-structural protein 2C co-localizes with VP1, 3A and 3D within these structures, but not with proteins associated with organelles of the host cell secretory pathway, including the ER, the ER to Golgi intermediate compartment (ERGIC), Golgi complex, trans-Golgi network (TGN) or lysosomes. The results suggest that these membranes of the secretory pathway are not used by the virus as a platform for replication and assembly, or that the FMDV-induced structures are formed by membrane rearrangements which involve exclusion of organelle-specific protein markers (Knox et al., 2005). 3D Enzyme As in other picornaviruses, the FMDV 3D protein is the viral-encoded RNA polymerase. The polymerase complex in FMDV-infected cells was first described over 30 years ago. This complex was originally called the FMD virus infection-associated antigen (FMD-VIAA), since it could be detected in infected cells by serum from FMD convalescent animals (Mason et al., 2003).<p> Elongation of the nascent RNA chains is catalyzed by 3Dpol, and poliovirus RNA replication takes place within a membranous replication complex consisting of non-structural proteins and RNA. Structures containing 3Dpol and RNA, resembling poliovirus replication complexes, have also been described in FMDV-infected cells (Mason et al., 2003). 3D Enzyme Elongation of the nascent RNA chains is catalyzed by 3Dpol, and poliovirus RNA replication takes place within a membranous replication complex consisting of non-structural proteins and RNA. Structures containing 3Dpol and RNA, resembling poliovirus replication complexes, have also been described in FMDV-infected cells (Mason et al., 2003). 3D Enzyme Following the initiation reaction, elongation of the minus strand begins, catalyzed by 3Dpol. For this to occur, the initiation complex must translocate to the 3' end of the plus-strand template. The mechanism by which this occurs is unknown, but one hypothesis suggests that binding of PABP to the poly(A) tract positions this region of the plus strand near the cre. The elongation of the nascent strands results in the formation of a double-stranded molecule, the replicative form (RF). Free minus strands are not detectable in vivo (Grubman and Baxt, 2004). 3D Enzyme The elongation of the plus strand by 3Dpol also occurs by an unknown mechanism (Grubman and Baxt, 2004). Genomic RNA in Replicative Complex Other Other After some rounds of translation, there has to be a switch so that translation of the viral RNA is stopped and RNA replication can commence, since these two processes appear incompatible on the same RNA molecule. Most picornaviruses replicate with high efficiency within susceptible cells, and within a few hours, the amount of viral RNA can represent 5% of the total RNA in cells (a level similar to that of all the cellular cytoplasmic mRNAs together). Nearly all of the FMDV RNA generated by replication is infectious; indeed, microinjection of cells with as little as 1 to 2 molecules of viral RNA is sufficient to initiate an infection. Thus, the replication of FMDV genomes within cells is remarkably efficient (Nayak et al., 2005).<p> Cytoplasmic structures staining for viral proteins form at perinuclear sites close to a dispersed Golgi apparatus. The viral non-structural protein 2C co-localizes with VP1, 3A and 3D within these structures, but not with proteins associated with organelles of the host cell secretory pathway, including the ER, the ER to Golgi intermediate compartment (ERGIC), Golgi complex, trans-Golgi network (TGN) or lysosomes. The results suggest that these membranes of the secretory pathway are not used by the virus as a platform for replication and assembly, or that the FMDV-induced structures are formed by membrane rearrangements which involve exclusion of organelle-specific protein markers (Knox et al., 2005).<p> The juxtanuclear location of FMDV replication complexes suggested an association with one or more Golgi compartments. In fact, when cells were double-stained with antibodies against 2C and Man II, which is found in the medial-Golgi, both proteins were found in the juxtanuclear region of the cell (Knox et al., 2005).<p> Initiation of minus-strand synthesis occurs in the cytoplasm in the presence of cellular mRNAs (Grubman and Baxt, 2004). Replicative form Cytoplasm Other Following VPg uridylylation, the viral 3' poly(A) tract serves as the initiation site for negative-strand RNA synthesis. The negative-strand viral RNAs then act as templates for the production of positive-strand RNA genomes that can be packaged into virions or can act as templates for the synthesis of viral proteins through subsequent rounds of cap-independent translation. Negative-strand RNA synthesis is thought to result in a double-stranded RNA intermediate that is termed replicative form or RF. A single negative-strand RNA can serve as a template for the production of several positive-strand RNA genomes, explaining the excess of positive-strand RNAs within an infected cell, with the ratio of positive to negative viral RNA strands being between 30:1 and 50:1 (Bedard and Semler, 2004).<p> The first step in picornavirus RNA replication is the synthesis of a minus-strand RNA molecule. This system has not been studied in FMDV; however, the models of RNA replication developed for poliovirus are probably quite similar. It is thought that translation of the plus-strand RNA must cease before minus-strand synthesis begins. The mechanism of this shutdown of translation of the plus strand is unclear (Grubman and Baxt, 2004).<p> Viral RNA synthesis begins with the production of negative-strand RNA from the genomic RNA template utilizing the VPg protein primer (Bedard and Semler, 2004).<p> Initiation of minus-strand synthesis occurs in the cytoplasm in the presence of cellular mRNAs (Grubman and Baxt, 2004). New Genomic Plus-strand RNA Other Other After formation of the RF, new plus-strand synthesis can begin (Grubman and Baxt, 2004).<p> For plus-strand synthesis to proceed, the RF must be unwound. The mechanism for this is also unclear. The picornavirus 2C protein both has ATPase activity and contains helicase motifs, but helicase activity has not been demonstrated. It has been shown that 2C and a cellular protein (p38) bind to the minus-strand 3' stem-loop, and this may act to destabilize the RF molecule. The possibility of involvement of either a cellular helicase or a nuclear protein has also been suggested, since the RF is infectious when transfected into whole cells but not when transfected into enucleated cells. The elongation of the plus strand by 3Dpol also occurs by an unknown mechanism (Grubman and Baxt, 2004). Virion Protomer Other The three large capsid proteins, VP1, VP0, and VP3, each fold into similar eight-stranded antiparallel beta-sheet barrels (Arnold et al., 1987).<p> The 3Cpro cleavage products of the P1 region are assembled into a protomer structure containing one copy of each of the proteins VP0, VP1, and VP3 (Grubman and Baxt, 2004). Virion Pentamer Other Five protomers can assemble into a pentamer (Grubman and Baxt, 2004).<p>Myristylation of 1AB is necessary for efficient assembly of capsid structures (Mason et al., 2003). Empty Capsid Other 12 pentamers assemble into the final capsid structure (Grubman and Baxt, 2004).<p> Five protomers assemble into a pentamer and twelve pentamers can assemble into an RNA-containing particle, called a provirion, or an empty capsid lacking the RNA genome (Mason et al., 2003).<p> The three-dimensional arrangements of the structural proteins within the virion provide the antigenic sites that elicit responses to vaccination or infection. In addition, these structures mediate binding to cell receptors, entry of the genome into cells, and determine the stability of the capsid to environmental factors. All of these functions influence how effectively the virus is able to spread within the host and between hosts, and hence are important molecular determinants of virulence. The cores of proteins 1B, 1C, and 1D consist of a highly conserved eight-stranded ?-barrel that is a hallmark of the capsid proteins of a large number of icosahedral viruses that affect both animals and plants. As with other picornaviruses, 1A, which appears to hold a structural conformation similar to N-terminal portions on 1C and 1D, is myristylated and buried within the virion. Therefore, the entire capsid surface is covered by portions of the other three proteins (Mason et al., 2003).<p> The N termini of VP1 to VP3 are usually confined to the interior of the capsid, whereas the C termini are located on the outside (Curry et al., 1997).<p> Although there have been a variety of studies to support the role of the empty capsids as intermediates in provirion assembly, more recent studies with an in vitro system programmed with viral RNA to synthesize poliovirus de novo, showed that only pentamer structures, not empty capsids, can interact with newly synthesized viral RNA to form virions. This suggests that the pentamers are the direct precursors of the provirion and that empty capsids may be a 'dead-end' product in the assembly reaction. However, it is possible that these empty capsid structures could serve as a storage form of pentamers for provirion assembly, which could help to reconcile the data of Yafal and Palma with a 'direct' assembly hypothesis (Mason et al., 2003). Provirion Other Five protomers assemble into a pentamer and twelve pentamers can assemble into an RNA-containing particle, called a provirion, or an empty capsid lacking the RNA genome. Assembly of the viral capsid and encapsidation of the viral RNA occur by mechanisms which are still obscure (Mason et al., 2003).<p>Two hypotheses have been advanced to explain the assembly of pentamers into provirions. The first postulates that pentamers assemble into empty capsids and the RNA is inserted to form the provirion, and the second postulates that pentamers interact directly with the RNA to form the provirion in the absence of an empty capsid intermediate. Support for the first hypothesis has come from studies on FMDV-infected cells indicating that radioactive label can be chased from protomers to pentamers to empty capsids to virions. In addition, poliovirus pentameric structures have been shown to self-assemble into empty capsids in vitro in the absence of viral RNA (Mason et al., 2003).<p>Although there have been a variety of studies to support the role of the empty capsids as intermediates in provirion assembly, more recent studies with an in vitro system programmed with viral RNA to synthesize poliovirus de novo, showed that only pentamer structures, not empty capsids, can interact with newly synthesized viral RNA to form virions. This suggests that the pentamers are the direct precursors of the provirion and that empty capsids may be a 'dead-end' product in the assembly reaction. However, it is possible that these empty capsid structures could serve as a storage form of pentamers for provirion assembly, which could help to reconcile the data of Yafal and Palma (1979) with a 'direct' assembly hypothesis (Mason et al., 2003).<p>The provirion, the last structural entity formed prior to generation of infectious virus, contains RNA surrounded by a capsid made up of proteins 1AB, 1C and 1D. Although provirions have been found in poliovirus-, hepatitis A-, and bovine enterovirus-infected cells, naturally occurring provirions have not been reported in FMDV-infected cells (Mason et al., 2003). Virion in Cytoplasm Other The final step in virion assembly is the maturation cleavage of VP0 into VP4 and VP2, requiring the presence of viral RNA. An aberrant cleavage of VP0 in FMDV empty capsids has been demonstrated, suggesting that viral RNA is required for the proper cleavage event to take place. Cleavage is thought to be autocatalytic and results from a conserved His residue in VP2 which activates local water molecules, leading to a nucleophilic attack on the scissile bond and cleavage. Maturation cleavage is required for the generation of infectious virus. In FMDV, site-directed mutations within VP0 led to the formation of noninfectious provirions that exhibited receptor binding and acid sensitivity, similar to the case for infectious virus. Upon acid dissociation, however, the generated pentamers were more hydrophobic than those from mature virions, suggesting that VP0 cleavage may be necessary for release of the RNA into the cytoplasm (Grubman and Baxt, 2004).<p>The final step in the transition of the provirion to the mature virion, is the cleavage of the 1AB protein to 1A and 1B, which occurs by unknown mechanisms and is dependent on the presence of the viral RNA. There has been a report of FMDV 1AB cleavage in empty capsids, however, this cleavage occurred at an abnormal site, suggesting that RNA is required for normal provirion maturation. These same studies suggested that the maturation cleavage mechanism involves a conserved His residue in 1B which activates local water molecules leading to a nucleophilic attack on the scissile bond resulting in 1AB cleavage. A similar model of cleavage has been predicted for other picornaviruses, and the model is intriguing, since the presence of RNA or an RNA-metal ion complex would increase the efficiency of the reaction. Regardless of the exact mechanism of the maturation cleavage, it is clear that the cleavage is autocatalytic (the provirion is not a stable intermediate) and is required for generation of infectious virus (Mason et al., 2003). Virion Extracellular Extracellular Other FMDV, like other members of the Picornaviridae, has a relatively short infectious cycle in cultured cells. Depending on the multiplicity of infection, newly formed infectious virions begin to appear at between 4 and 6 h after infection (Grubman and Baxt, 2004). eIF4G Ribosome Nucleic acid binding Initiation of FMDV RNA translation requires only the Lpro-generated C-terminal eIF4G cleavage product, which binds to the FMDV IRES and interacts with 40S ribosomal subunit-bound eIF4A and eIF3. In addition, eIF4B is bound to the FMDV IRES and has been identified in both 48S preinitiation complexes and 80S ribosomes (Grubman and Baxt, 2004).<p> A key role in the process of internal initiation is attributed to eIF4G. It appears to take on the role of a multipurpose adapter that connects the RNA with the ribosome. The N-terminal domain of eIF4G that interacts with the cap-binding protein eIF4E is clipped off by the proteases of several picornaviruses. The C-terminal domain interacts with the ribosome-bound eIF3 and the RNA helicase eIF4A and is sufficient to confer internal translation initiation of several picornaviruses. Cleavage of eIF4G even stimulates translation from the type I IRES elements of entero-/rhinoviruses, whereas the FMDV and other type II IRES elements work well with the cleaved or the uncleaved form of eIF4G (Saleh et al., 2001). PTBP Cytoplasm Nucleic acid binding The FMDV IRES interacts with a number of cellular proteins, including initiation factors important for normal cellular mRNA translation. A host factor of 57 kDa, subsequently identified as the nuclear polypyrimidine tract binding protein (PTBP), was shown to interact with at least two regions of the IRES. Deletion of these two sites inhibited both the binding of the protein and in vitro translation (Grubman and Baxt, 2004).<p> A cellular 57-kDa protein (p57) that binds specifically to the internal translation initiation site in the 5' untranslated region of foot-and-mouth disease virus RNA was detected in cell extracts of different mammalian species by UV cross-linking. The protein binds to two distinct sites of the translation control region which have as the only common sequence a UUUC motif. The first binding site consists of a conserved hairpin structure, whereas the second binding site contains an essential pyrimidine-rich region without obvious secondary structure. Competition experiments indicate that the complexes with the two binding sites were formed by a single p57 species. The protein binds also to the 5' untranslated region of other picornaviruses (Luz and Beck, 1991).<p> A cytoplasmic 57-kDa protein (p57) that bind to picornavirus IRES elements was thus found to be an essential trans-acting factor for their translation. We report here that p57 is identical to the 'pyrimidine tract-binding protein' (PTB), also know as heterogenous nuclear ribonuclearprotein I (hnRNP I). This polypeptide has been implicated in various nuclear processes involving pre-RNA (Hellen et al., 1993). ITAF(45) Nucleic acid binding More recently a second host factor, which is required for FMDV IRES-driven translation but not for translation of cardiovirus mRNA, has been identified. This 45-kDa protein, IRES-specific trans-acting factor (ITAF45), along with PTBP, is required for the formation of the 48S translation-initiation complex (Grubman and Baxt, 2004). PCBP-hypothetical link- Nucleic acid binding A third host factor, PCBP, which is required for translation of poliovirus RNA has not to date been shown to be involved in FMDV translation. However, the presence of the poly(C) tract upstream of the IRES suggests that it may also play a role in FMDV translation, genome replication, or both. It has also been postulated that PCBP facilitates a circularization of the poliovirus genome to modulate the balance between translation and RNA replication (Grubman and Baxt, 2004). Recently it has been shown that a genetically engineered FMDV, which is unable to perform the maturation cleavage of VP0 to VP2 and VP4 is noninfectious, can adsorb to cells in culture, and is acid sensitive. Thus, the breakdown of 140S virus to pentameric subunits by itself does not lead to productive infection, but there must be other events after the breakdown. These results indicate that the viral receptor is responsible only for docking the virus to the membrane of the susceptible cell and plays no role in viral uncoating, which is consistent with the ability of FMDV to utilize multiple receptors for infection in cell culture (Grubman and Baxt, 2004). In general, picornaviruses utilize endocytic pathways, which are either clathrin mediated, caveola mediated, or lipid raft dependent, to enter cells after binding to their receptors (O'Donnell et al., 2005). <p> In order to determine the endocytic pathway used by FMDV to enter the cell, the distribution of virus with markers for the clathrin or caveola pathway was examined. After adsorption at 4 C, virus appears to colocalize with clathrin as early as 5 min after the temperature is shifted to 37 C. By 30 min after the temperature shift, little or no colocalization of virus and clathrin can be observed, which probably is the result of the uncoating of clathrin from the clathrin-coated pit after separation from the plasma membrane. In addition, virions did not colocalize with caveolin-1, a marker for the caveola-mediated endocytosis pathway, during the internalization process. To verify these results biochemically, we utilized chlorpromazine, a member of a class of compounds that inhibits the formation of clathrin-coated pits and causes pits to disappear from the cell surface. In the presence of this drug, viral infection was markedly inhibited. Finally, viral infection was not inhibited in the presence of nystatin, a cholesterol-sequestering and lipid-raft disrupting compound. Clathrin-mediated endocytosis is generally not dependent on lipid rafts (O'Donnell et al., 2005). By using a series of lysosomotropic agents, which raise the pH of intracellular endosomes, it has been demonstrated that the virus probably breaks down upon entering an acidic endosome (Grubman and Baxt, 2004).<p> FMDV enters cells via a mechanism of receptor-mediated endocytosis in which the low pH of the endosomal compartment triggers uncoating of the viral genome. FMDV, unlike other picornaviruses, uncoats in a single step, which is inhibited by lysosomotropic agents, such as chloroquine and ammonium chloride (Wachsman et al., 1998).<p> Virus disassembly proceeds directly to its 12S pentameric subunits, RNA and VP4. FMDV is unusual among the picornaviruses, as its capsid is extremely sensitive to acid and dissociates at a pH just below neutrality. Within the capsid, the interpentamer contacts are largely ionic, and the trigger for capsid disassembly is believed to be the protonation of specific histidine residues at the pentamer boundary (Berryman et al., 2005). Following uncoating, the RNA is released into the cytoplasm by an as-yet-unknown mechanism and begins a round of viral translation (Grubman and Baxt, 2004). Following uncoating, the RNA is released into the cytoplasm by an as-yet-unknown mechanism and begins a round of viral translation. The genome-linked protein VPg is cleaved by a cellular enzyme prior to translation of the incoming RNA; however, protein synthesis initiation complexes can be formed with mRNA containing VPg (Grubman and Baxt, 2004). Initiation of FMDV RNA translation requires only the Lpro-generated C-terminal eIF4G cleavage product, which binds to the FMDV IRES and interacts with 40S ribosomal subunit-bound eIF4A and eIF3. In addition, eIF4B is bound to the FMDV IRES and has been identified in both 48S preinitiation complexes and 80S ribosomes. The FMDV IRES interacts with a number of cellular proteins, including initiation factors important for normal cellular mRNA translation. A host factor of 57 kDa, subsequently identified as the nuclear polypyrimidine tract binding protein (PTBP), was shown to interact with at least two regions of the IRES. Deletion of these two sites inhibited both the binding of the protein and in vitro translation. More recently a second host factor, which is required for FMDV IRES-driven translation but not for translation of cardiovirus mRNA, has been identified. This 45-kDa protein, IRES-specific trans-acting factor (ITAF45), along with PTBP, is required for the formation of the 48S translation-initiation complex. A third host factor, PCBP, which is required for translation of poliovirus RNA has not to date been shown to be involved in FMDV translation. However, the presence of the poly(C) tract upstream of the IRES suggests that it may also play a role in FMDV translation, genome replication, or both. It has also been postulated that PCBP facilitates a circularization of the poliovirus genome to modulate the balance between translation and RNA replication (Grubman and Baxt, 2004).<p> The RNA codes for only one long open reading frame. Translation proceeds primarily from a single strong initiation site and produces a giant precursor polyproteins (M(r) approximately 250,000) that is divided into three regions: P1, P2, and P3 (Arnold et al., 1987). The very first cleavage within a polyprotein takes place while the polypeptide is still nascent on a ribosome (Arnold et al., 1987). <p> Unlike enteroviruses and rhinoviruses, the primary cleavage event of aphthoviruses is at the L-P1 junction releasing the L protein from the N-terminus of the viral polyprotein. For aphthoviruses, this primary cleavage occurs in cis by the L proteinase which cleaves at its own carboxy terminus (Bedard and Semler, 2004).<p> The FMDV capsid precursor P1-2A is released from the polyprotein by cleavage at its N-terminus by the L protease and at its C-terminus by the 2A protein. Breakage of the viral polyprotein at the 2A/2B junction is very rapid within cells and within in vitro translation reactions, and no uncleaved species are detected (Belsham, 2005).<p> The FMDV P3 precursor is separated from P2 and processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (3VPg), 3C protease and 3D RNA polymerase plus a variety of intermediates (e.g. 3CD) (Belsham, 2005). The P1/2A cleavage to form the structural protein precursor, P1 is performed by the 3C protease (Fry et al., 2005).<p> Based on these analyses, genetic studies were performed to examine the potential active site residues of FMDV 3Cpro. To this end, WT FMDV 3Cpro proteins were expressed in E. coli and tested for their ability to cleave synthetic substrates corresponding to viral polyprotein precursors. These studies revealed that P1-2A cleavage occurs much more rapidly than cleavage within P2. In addition, studies performed with E. coli-expressed mutant forms of 3Cpro suggested that Cys163 was the catalytic nucleophile, and that His46 and Asp84 also formed part of the catalytic triad. Additional support for the above model of the active site of FMDV 3Cpro has been provided by X-ray crystallography of HRV and hepatitis A virus 3Cpro (Mason et al., 2003). The P1-2A precursor is processed by the 3C protease to yield VP0 (1AB), VP3 (1C) and VP1 (1D). These are the components of natural empty capsids and 60 copies of each protein will self-assemble to form particles (Belsham, 2005).<p> The proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C (Vakharia et al., 1987). The FMDV 2BC precursor is processed to 2B and 2C by the 3C protease (Belsham, 2005). The FMDV P3 precursor is separated from P2 and processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (VPg), 3C protease and 3D polymerase plus a variety of intermediates (e.g. 3CD) (Belsham, 2005). The FMDV P3 precursor is separated from P2 and processed by the 3C protease to 3A, the three distinct copies of the 3B peptide (VPg), 3C protease and 3D polymerase plus a variety of intermediates (e.g. 3CD) (Belsham, 2005). To replicate the positive-sense genome, an antisense RNA has to be synthesized which then functions as the template for the production of new positive-sense infectious genomes. RNA is synthesized by the viral 3D protein that functions as an RNA-dependent RNA polymerase and will be referred to as 3Dpol. Interestingly, 3Dpol requires the uridylylated form of the 3B/VPg peptide (VPgpU or VPgpUpU) to act as the primer for both positive- and negative-strand synthesis. In recent years, the mechanism involved in the synthesis of this modified peptide primer has become clearer (Nayak et al., 2005).<p> Cytoplasmic structures staining for viral proteins form at perinuclear sites close to a dispersed Golgi apparatus. The viral non-structural protein 2C co-localizes with VP1, 3A and 3D within these structures, but not with proteins associated with organelles of the host cell secretory pathway, including the ER, the ER to Golgi intermediate compartment (ERGIC), Golgi complex, trans-Golgi network (TGN) or lysosomes. The results suggest that these membranes of the secretory pathway are not used by the virus as a platform for replication and assembly, or that the FMDV-induced structures are formed by membrane rearrangements which involve exclusion of organelle-specific protein markers (Knox et al., 2005). Following the initiation reaction, elongation of the minus strand begins, catalyzed by 3Dpol. For this to occur, the initiation complex must translocate to the 3' end of the plus-strand template. The mechanism by which this occurs is unknown, but one hypothesis suggests that binding of PABP to the poly(A) tract positions this region of the plus strand near the cre. The elongation of the nascent strands results in the formation of a double-stranded molecule, the replicative form (RF). Free minus strands are not detectable in vivo (Grubman and Baxt, 2004). For plus-strand synthesis to proceed, the RF must be unwound. The mechanism for this is also unclear. The picornavirus 2C protein both has ATPase activity and contains helicase motifs, but helicase activity has not been demonstrated. It has been shown that 2C and a cellular protein (p38) bind to the minus-strand 3' stem-loop, and this may act to destabilize the RF molecule. The possibility of involvement of either a cellular helicase or a nuclear protein has also been suggested, since the RF is infectious when transfected into whole cells but not when transfected into enucleated cells. The elongation of the plus strand by 3Dpol also occurs by an unknown mechanism (Grubman and Baxt, 2004). The mechanisms of encapsidation and maturation are still unresolved and are probably the least studied of all of the steps in the replication cycle. Again, most of the studies have been done with the enteroviruses, and therefore analogies must be drawn with FMDV. In broad terms, the 3Cpro cleavage products of the P1 region are assembled into a protomer structure containing one copy of each of the proteins VP0, VP1, and VP3 (Grubman and Baxt, 2004). During assembly, five protomers, each containing one copy of VP0, VP1, and VP3, assemble into a pentamer, and 12 pentamers associate with a newly transcribed RNA molecule to form a virus particle (Curry et al., 1997). The assembly and encapsidation mechanism is unclear; either the pentamers assemble into empty capsids and the RNA [covalently linked to VPg (3B)] is inserted to form the provirion (the immature particle in which the maturation cleavage of VP0 has yet to occur) or the pentamers interact directly with the RNA/VPg, forming the provirion without an empty capsid intermediate (Fry et al., 2005). A number of intermediate particles have been identified in picornavirus-infected cells, including protomers, pentamers, a particle containing RNA with an uncleaved VP0 (provirion), and a particle with an uncleaved VP0 lacking RNA (empty capsid). Two unresolved issues in picornaviral maturation are what signals are necessary for encapsidation of the RNA and what are the roles of the empty capsid and provirion (Grubman and Baxt, 2004).<p> The assembly and encapsidation mechanism is unclear; either the pentamers assemble into empty capsids and the RNA [covalently linked to VPg (3B)] is inserted to form the provirion (the immature particle in which the maturation cleavage of VP0 has yet to occur) or the pentamers interact directly with the RNA/VPg, forming the provirion without an empty capsid intermediate (Fry et al., 2005). The final step in the transition of the provirion to the mature virion, is the cleavage of the 1AB protein to 1A and 1B, which occurs by unknown mechanisms and is dependent on the presence of the viral RNA (Mason et al., 2003).<p> Cleavage is thought to be autocatalytic and results from a conserved His residue in VP2 which activates local water molecules, leading to a nucleophilic attack on the scissile bond and cleavage. Maturation cleavage is required for the generation of infectious virus. In FMDV, site-directed mutations within VP0 led to the formation of noninfectious provirions that exhibited receptor binding and acid sensitivity, similar to the case for infectious virus. Upon acid dissociation, however, the generated pentamers were more hydrophobic than those from mature virions, suggesting that VP0 cleavage may be necessary for release of the RNA into the cytoplasm (Grubman and Baxt, 2004).<p> Following encapsidation of the RNA, the maturation cleavage reaction (VP0 to VP2 and VP4) takes place (Grubman and Baxt, 2004). TEXT Foot-and-Mouth-Disease virus Foot-and-mouth disease virus (FMDV) is the prototype member of the Aphthovirus genus of the family Picornaviridae (Mason et al., 2003). In the mature virus, the genome is encapsidated in an icosahedral structure composed of 60 copies of four proteins (1A, 1B, 1C, and 1D). The genome contains a single long open reading frame (ORF), that has two alternative initiation sites, and the encoded polyprotein can be processed into over a dozen well-described mature polypeptides as well as a variety of partial cleavage intermediates (Mason et al., 2003). 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