Picornaviruses are small positive strand RNA viruses with single stranded genomic RNA capable of causing various diseases in humans (Porter, 1993). The picornavirus RNA genome has 3' poly(A) tail and a virus encoded particle Vpg at the 5' end. Unlike other RNA genome viruses, picornavirus doesn't contain a 7- methyl guanosine cap at the 5'end intead they contain VPg a viral protein linked at the 5' end of the genome (Bedard and Semler, 2004). IRES (Internal ribosome entry site) is a clover leaf secondary structure at the 5' non-coding region and in the polypeptide there is a 3' noncoding region which contains the poly(A) tract required for the synthesis of minus RNA strand for RNA replication and translation. The minus strand RNa increases the efficiency of RNA replication and translation. The structural and non structural proteins are found at the polyprotein and the polyprotein is divided into three regions such as P1, P2, P3 where P1 contains the structural proteins (VP1, VP2, VP3 and VP4) required for capsid formation and the non structural proteins P2 and P3 are required for replication and membrane rearrangement. The P2 and P3 region contains proteinase 3C and viral RNA dependent polymerase 3D (Stanway, 1990).
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Figure1: The polyprotein products and their major functions. There are 11 mature polypeptides with three main cleavage intermediates. UTR- Untranslated region; IRES- Internal ribosome entry site; VPg- Viral protein genome-linked (Lin et al, 2009).
Figure2: Life cycle of Picornavirus (Whitton et al, 2005)
Processing of proteins
The processing of proteins starts from the primary cleavage occurring between P1 and P2, mediated by viral protease 2A containing the cysteine nucleophile and it cleaves P1 and P2 leaving the viral polyprotein at cis. The P2 and the P3 precursors are separated from P1 region. For aphthovirus self cleavage takes place at the P1 domain region of the polyprotein by L proteinase at the cis whereas the L proteins in cardiovirus possess no proteolytic activity and hence in aphthovirus the cleavage of P1 is initiated by proteinase 3C. 2A proteinase also cleaves the cellular factors in entero and rhinovirus where eIF4G, a cap binding complex is cleaved and due to this even the there is a shut off in host translation. The L proteinase also cleaves eIF4G in aphtho virus during primary cleavage (Ryan and Flint,1997). There are four structural proteins in Picornavirus capsids such as VP1, VP2, VP3 and VP4 where the VP4 protein is inside of the capsid. The VP4 proteins are formed by the cleavage of VP0 precursor, late during the assembly and are modified after translation by the covalent attachment of myristic acid at the amino terminus (Cann, 1997).
Picornaviruses consist of three types of proteinase L, 2A and 3C. the aphtho or F M D V polyproteins are processed by more than one proteinase. The L proteinase are available in two forms Lb pro and Lab pro. L pro possess the same function as 2A proteinase of entero and rhinovirus which cleave the host cell protein eIF4G but the major difference between L pro and 2A is that the L pro cleave in between Gly479 and Arg470 residues whereas 2A cleave in between Arg486 and Gly487. Lb pro plays a major role in substrate binding and also in shut off host cell translation like that of 2A proteinase (Ryan and Flint,1997).
2A proteinase cleaves at its own N terminus and the primary cleavage is carried at the P1 capsid protein precursor. The nature of 2A proteinase is unclear and the sequence similarities led to the understanding that 2A pro catalytic triad composed of His20, Asp38 and also an active site nucleophile of cysteine. 2A pro can be inhibited by active thiol proteinases such as iodoacetamide and N-ethylmaleimide. 2A proteinase also inhibits the host cell protein synthesis which mediated the cleavage of eIF-4G, a 220K Da polypeptide but it was later understood that the 2A pro just initiated as an activating factor for another proteolytic activity to cleave eIF-4G. Generally 2A pro shuts off the host cell translation because eIF-4G deals with cap dependent mode of translation in host cell. In poliovirus, 2A acts a trans activator of translation at IRES when host cell is not imhibited. When 2A pro was mutated it led to loss of cleavage activity in trans but not in cis, and no replication of viral RNA was seen hence this confirms that 2A pro is required for viral RNA replication. The aphtho and cardiovirus 2A proteinase show no sequence similarity to entero/rhino virus although 2A protease are similar in size.
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The primary cleavage of hepatovirus and echovirus does not take place by the cleavage of 2A proteinase and the 2A protein in hepatovirus and echovirus showed no proteolytic activity (Ryan and Flint,1997). 2B and its precursor 2BC is a viral protein consisting of two hydrophobic regions with α amphipathic a-helix domains leading to the alteration of membranes in the infected cells. The virus induced vesicles are formed when 2B and its precursor 2BC enter into the host membrane of Golgi and ER complex by altering the permeability of plasma membrane to form virporin complex. Once the 2B and the precursor 2BC enters the host there is an imbalance in Ca2+ homeostatis mechanism and blocks protein transport from ER to Golgi and also initiates anti-apoptosis property. The 2B protein also blocks the activation of IRF-3 in Hepatitis A virus through which the cellular IFN-β gene transcription is inhibited so that there is no harm to Hepatitis A virus in the host. (Lin et al, 2009). The viral RNA was anchored for the spatial arrangement required for replication by the 2C protein. The mutagenesis experiments confirmed that the 2C proteins are involved in strand separation of viral RNA while replication (Porter, 1993)
The secondary cleavage is carried out within the viral proteins and hence it is mediated by 3C proteinase which plays a very important role in protein processing and RNA replication. The replication proteins are generated within the P2 and P3 precursor proteins when 3C self cleaves at the P3 region of the polyprotein. 3C proteinase or the 3CD precursors cleaves the poly(A) binding protein to inhibit viral translation during late poliovirus infection. 3C also cleaves the host cell protein required for transcription. The key processing step for the viral protein processing cascade is initiated by 3C proteinase. The 3CD proteins also play a major role in carring out important functions during RNA replication (Bedard and Semler, 2004).
The major function of 3C proteinase in secondary cleavages is that it process the capsid and the replicative protein precursors. The processing of capsid in poliovirus is done by 3CD proteinase and not 3C proteinase. The 3C proteins are also used to cleave various number of host cell proteins such as histone H3, transcription factor IIIC, TATA binding protein and microtubule-associated protein 4. The 3CD proteinase depends on the host cell protein EF-1 α and the host cell factor is replaced by 3CD forming 3AB:3CD proteinase complex to bind at the 3' end of the poliovirus genome (Ryan and Flint, 1997). The 3A protein inhibits the cellular protein function and also presents the membrane proteins during viral infection. When the poliovirus 3A protein was mutated, the uridlylation of VPg was affected and also inhibited the viral RNAs plus strand initiation (Porter, 1993).
2B is a viral protein which is required for virion release by altering the cell membrane increasing permeability which is required for poliovirus RNA replication. 2C proteins and its precursor 2BC is necessary for the re-arrangement of intracellular membranes and also for the viral induced cytoplasmic vesicles. 2C binds with the minus strand of poliovirus RNA at the 3' non coding region and hence it plays a role in positive RNA viral strand synthesis and also in minus strand RNA synthesis. 3C and 3D protein helps in immune response interference and also in viral RNA replication. 3A protein is very much important because these proteins help the picornaviruses to escape from MHC-I ( Major histo compatability) expression and intracellular membrane transport by inhibiting both the MHC-I and intracellular membrane transport of the host cell. 3B protein also called as VPg is linked to the 5' end of both the positive and negative strand RNAs. 3AB, 3C, 3CD and 3D are required during the process of assembly in replication and also initiates viral RNA polymerase 3D and self cleavage of 3CD. 3C and 3D are involved in binding viral RNA, protein processing and RNA replication. The cloverleaf structure of poliovirus virus and coxsackievirus consist of stem loops in which the viral polymerase precursor, 3CD binds to the stem loop I. 3CD also binds with the host cell protein poly r(C) binding protein 2 (PCBP2) to help only in RNA replication. 3D is responsible for VPg uridylylation and RNA chain elongation while synthesising viral RNA because it contains RNA dependent polymerases and even tends for error prone and mis-incorporation of 1-2 nucleotides per replication (Bedard and Semler, 2004).
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VP1, VP2 and VP3 are the three larger capsid proteins folded into eight stranded antiparallel β barrels and a small fourth protein called VP4 is located inside the capsid. The 3C protease cleaves VP3, VP1 and VP0 at the P1 region. The amino termini of VP0, VP3, VP1 initiates the assembly of the virion particle. VP0 peptides are cleaved into VP2 and VP4 at the final stage of processing and assembly. VP4 and VP2 are adjacent to each other at their ends when cleaved. The infection of the host is initiated when the capsid proteins bind to the receptor on the host membrane. ICAM-1 (intercellular adhesion molecule 1) is the receptor molecule for the major rhinovirus which binds the cell to adjacent substrates. Poliovirus receptor molecule is an integral membrane protein consisting of one variable and two constant domains and this receptor molecule attaches to the host cell to initiate replication process. The general receptors used by the various picornaviruses are poliovirus receptor for poliovirus attachment, ICAM-1 receptor for major rhinovirus, LDL-R for minor group rhinovirus, CD55 or DAF receptor for some echoviruses and group B coxsackie B1-B6 receptors. A deep cleft known as canyon is formed by flanking the monomers, VP1, VP2 and VP3 which helps the virus to escape the immune response by the host cell. The interaction of capsid proteins with the intracellular host factors affects the induction of apoptosis (Lin et al, 2009).
Host cell shut off mechanism
The mRNA of picornavirus is uncapped and hence the translation takes place by directly introducing ribosomes at internal ribosome entry sites (IRES). The cleavage of eukaryotic translation initiation factor, eIF4G by 2A protease inhibits the cap dependent mRNA translation of the host cell. When poliovirus is introduced into the host it inhibits the host cell translation leading to apoptotic cell death. The eIFGII is more resistant to the infection of poliovirus than eIFGI. The death inducing proteins encoded by cellular mRNA were translated by cap independent translation leading to apoptotic death. The cleavage of eIF4GI by caspase 3 also induces apoptotic cell death but differs from poliovirus 2A protease process. Severe inhibition of translation leading to apoptosis is seen by both the caspase 3 activity and 2A protease. IRES elements in mRNAs encode proteins which regulate apoptotis. Even poly (A) protein and dystropin protein are also cleaved by 2A protease.. Hence, the cleavage of poly(A) binding protein by 2A protease cause apoptotis and the cleavage of dystrophin protein can induce apoptotic process due to cytoskeleton disruption (Goldstaub et al, 1999).
The enterovirus 2B protein suppresses apoptotic pathway of the host cell by controlling intracellular Ca2+ homeostatis. The apoptotic responses are initiated by the 3C and 2A proteinase to inhibit cellular transcription and cap dependent translation. When the 2B proteins are suppressed by caspase-3 activation it leads to apoptotis (Campanella et al, 2004). The cellular mRNAs encode for the death inducing proteins which are translated by cap independent translation. When eIF4GI and eIF4GII are cleaved by 2A protease it leads to apoptotic death because it inhibits cap dependent translation. The 2A proteins also cleave the poly(A) binding protein and the dystropin protein which leads to apoptotis through a translational mechanism (Goldstaub et al, 2000).
The eIFE is the component of cap binding complex of cap structure at 5' end of mRNA. The 40 S ribosomal subunit checks at the 5' non coding region until it finds the initiating codon or the the authentic start codon AUG and then sends a signal to the 60S to form a complex. For translation to occur in mRNA the 5' non coding region with the cap end should bind with the eIFE and once it binds to the cap the 40 S ribosomal subunit scans for the authentic initiation or start codon AUG and once it finds the AUG codon it gives a signal to 60 S ribosomal subunit to form a complex with 40 S and initiate the translation along with initiation factors (Bedard and Semler, 2004).
In picornavirus the 5' end of mRNA in the non coding region is not capped and hence to initiate translation, the cap independent mechanism is required. Hence in picornavirus, the 40 S ribosomal subunit scans for ribonucleo protein complex at 5' non coding region and initiation take place to recognise authentic start codon. The eIF4G is cleaved by viral proteinase 3C and 2A which shuts off the host cell translation (cap dependent) and also cleaves poly(A) binding protein (PABP)and hence it inhibits the host cell translation.
When the host cell is infected by Foot and mouth disease virus (FMDV), the eIF4G subunit which is responsible for the cap dependent translation of the host is cleaved by the L protein and for the Encephalomyocarditis virus (EMCV), the cap dependent translation of host is blocked by a repressor protein 4E-BP1 which binds at the 5' cap region and hence the repressor protein 4E-BP1 blocks the binding of eIF-4E which is the cap binding subunit and hence the host translation is shut off. The Hepatitis A virus (HAV) does not shut off the host translation because it requires eIF4G for its translation (Bedard and Semler, 2004).
All picornavirus consist of internal ribosome entry site (IRES) and are mapped to the 5' non coding region. There are four types of IRES seen in picornaviruses based on the RNA secondary structures. The enterovirus and rhinovirus comes under the Type I IRES. Aphthovirus and cardiovirus comes under the Type II IRES and the Hepatitis virus comes under the Type III IRES. The porcine reschovirus comes under the Type IV elements.
The IRES mediated translation is initiated by two factors
Canonical initiation factors and
IRES trans-activating factors.
Canonical initiation factors to initiate IRES mediated translation:
The IRES elements of poliovirus and EMCV are similar and require the initiation factors to be primed at the 40S ribosomal subunit. The IRES mediated translation is initiated by certain canonical factors such as eIF4G and eIF4B to bind to the viral RNA and also certain other subunits such as eIf3 and eIF2 to pre-bind to the 40 S ribosomal subunits. The IRES translation of poliovirus and EMCV is promoted by the poly(A) binding protein (Lin et al, 2009).
Figure Structural feature of IRES elements (Lin et al, 2009)
Noncanonical initiation factors to initiate IRES mediated translation:
The polypyrimidine tract-binding protein (PTB) is a 57 KDa mRNA splicing factor which increases and promotes the activity of IRES in poliovirus. The molecular switching from translation to the replication of poliovirus is done by the proteolytic cleavage of PTB. The PTB also functions as RNA chaperon by stabilizing the type II IRES of FMDV and EMCV. Lupus autoantigen (La) is a 52 KDa of nuclear protein which binds to certain distinct parts of HAV IRES and with small interfering RNA and hence the HAV IRES translation and replication is suppressed. Poly(rC) binding protein (PCBP2)bind to the type I IRES of picornavirus and hence it leads to internal initiation of translation in type I IREs elements. The heterogenous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA binding protein which binds to the 5' UTR of HRV2 and regulates transcription (Lin et al, 2009).
The picornavirus infection has lowered the level of host cell transcription and hence there is a increase in the number of viral RNA molecules. The nuclear localization signal (NLS) present in the poliovirus 3D protein targets the 3CD precursor to the nucleus for autocatalytic final maturation and allow the release of 3C protease (Bedard and Semler, 2004).)
Picornavirus RNA replication:
The new positive and negative strand viral RNA are synthesised by using the viral encoded RNA dependent RNA polymerase 3D. The RNA dependent RNA polymerase 3D acts as a protein primer and forms VPg-pU-pU which initiates the viral RNA replication and the process is known as VPg uridylylation. When VPg uridylylation is done, the poly(A) tract at the 3' end acts as an initiation site for the synthesis of negative strand RNA synthesis. The negative strand developed act as a template for the synthesis of new viral positive RNA strand by cap independent translation. Many or numerous copies of positive viral RNA strand can be synthesised from a single negative strand.
In picornaviruses, there are numerous RNA sequences and secondary structures within the 5' non coding region which are essential for the RNA replication. The viral protein 3CD and the host protein, PCBP binds at the 5' cloverleaf structure of the non coding region. The cellular host protein PCBP binds to loop b and the 3CD binds to loop d of the 5' cloverleaf structure. A ternary complex is formed when 3CD and PCBP2 binds with the cloverleaf structure along with the viral RNA to form RNA replication. When PCBP2 was depleted, there was poor RNA synthesis in poliovirus and hence it was recognised that PCBP2 play a vital role in RNA replication. PABP is a cellular protein which binds to the poly(A) tract at the 3' end of viral RNA interact with 5' cloverleaf structure of PCBP2 and the viral protein 3CD. When the PABP at the 3' end interact with PCBP2 at the 5' end, the viral RNA may interact and hence there is an initiation of replication process. Even the 3AB and 3CD proteins interact at the 5' cloverleaf structure to each other there by initiating viral RNA replication. The cis-acting replication element (cre) was found within the coding region of picornavirus genomic RNA which is required for viral replication and viability. In aphthovirus the cre structure is at the non coding region of the 5' end. The cre sequence AAACA was found in the conserved hairpin structure at the coding region of picornaviruses required for RNA replication. The cre elements act as a binding site for viral replication proteins and also as a template for VPg uridylylation. At cre element, VPg is more efficient than at the poly(A) tract and uridylylation on cre structure leads to only positive strand RNA synthesis and for uridylylation and replication initiation of the minus strand uses the poly(A) tract. The interaction between 3CD with cre and cloverleaf RNA may arrange the viral RNA structurally for efficient RNA replication. The 3' NCR and the 3' poly(A) tract forms the site of replication for minus strand RNA synthesis. The poly(A) tract at the 3' end functions for virus viability, impart stability and also for the efficient RNA replication by interacting with viral RNA at the 5' end (Bedard and Semler, 2004).
The negative strand is first formed by using the VPg protein primer and the VPg plays an important role in replication. The enzymatic activity of 3D polymerase has lead to VPg uridylylation which covalently couples with the tyrosine residue in the VPg protein. The 3' poly (A) tract template may also involve other host proteins for replication. The 2C bind at the 3' end the negative strand showing ATPase activity. The RNA polymerase 3D unwinds to develop viral RNA synthesis (Bedard and Semler, 2004).