Picornaviruss are little positive strand RNA viruses with individual stranded genomic RNA capable of doing assorted diseases in worlds ( Porter, 1993 ) . The picornavirus RNA genome has a 3 ‘ poly ( A ) tail and a virus encoded atom Vpg at the 5 ‘ terminal. Unlike other RNA genome viruses, picornavirus does n’t incorporate a 7- methyl guanosine cap at the 5’end intead they contain VPg a viral protein linked at the 5 ‘ terminal of the genome ( Bedard and Semler, 2004 ). IRES ( Internal ribosome entry site ) is a trefoil foliage secondary construction at the 5 ‘ non-coding part and in the polypeptide there is a 3 ‘ noncoding part which contains the poly ( A ) piece of land required for the synthesis of subtraction RNA strand for RNA reproduction and interlingual rendition. The subtraction strand RNa increases the efficiency of RNA reproduction and interlingual rendition. The structural and non structural proteins are found at the polyprotein and the polyprotein is divided into three parts such as P1, P2, P3 where P1 contains the structural proteins ( VP1, VP2, VP3 and VP4 ) required for mirid bug formation and the non structural proteins P2 and P3 are required for reproduction and membrane rearrangement. The P2 and P3 part contains proteinase 3C and viral RNA dependant polymerase 3D ( Stanway, 1990 ).
Figure1: The polyprotein merchandises and their major maps. There are 11 mature polypeptides with three chief cleavage intermediates. UTR- Untranslated part ; IRES- Internal ribosome entry site ; VPg- Viral protein genome-linked ( Lin et al, 2009 ).
Processing of proteins
The processing of proteins starts from the primary cleavage happening between P1 and P2, mediated by viral peptidase 2A incorporating the cysteine nucleophile and it cleaves P1 and P2 go forthing the viral polyprotein at Commonwealth of Independent States. The P2 and the P3 precursors are separated from P1 part. For aphthovirus ego cleavage takes topographic point at the P1 sphere part of the polyprotein by L protease at the Commonwealth of Independent States whereas the L proteins in cardiovirus possess no proteolytic activity and hence in aphthovirus the cleavage of P1 is initiated by protease 3C. 2A protease besides cleaves the cellular factors in entero and rhinovirus where eIF4G, a cap-binding composite is cleaved and due to this even there is a shut off in host interlingual rendition. The L protease besides cleaves eIF4G in aphtho virus during primary cleavage ( Ryan and Flint,1997 ). There are four structural proteins in Picornavirus mirid bugs such as VP1, VP2, VP3 and VP4 where the VP4 protein is inside of the mirid bug. The VP4 proteins are formed by the cleavage of VP0 precursor, tardily during the assembly and are modified after interlingual rendition by the covalent fond regard of myristic acid at the amino end point ( Cann, 1997 ).
Picornaviruses consist of three types of protease L, 2A and 3C. the aphthous or F M D V polyproteins are processed by more than one protease. The L protease are available in two signifiers Lb pro and Lab pro. L pro possess the same map as 2A protease 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 function in substrate binding and besides in shut off host cell interlingual rendition like that of 2A protease ( Ryan and Flint,1997 ) .
2A protease cleaves at its ain N end point and the primary cleavage is carried at the P1 mirid bug protein precursor. The nature of 2A protease is ill-defined and the sequence similarities led to the apprehension that 2A pro catalytic three composed of His20, Asp38 and besides an active site nucleophile of cysteine. 2A pro can be inhibited by active thiol proteases such as iodoacetamide and N-ethylmaleimide. 2A protease besides inhibits the host cell protein synthesis which mediated the cleavage of eIF-4G, a 220K Da polypeptide but it was subsequently understood that the 2A pro merely initiated as an energizing factor for another proteolytic activity to split eIF-4G. By and large 2A pro shuts off the host cell interlingual rendition because eIF-4G trades with cap dependent manner of interlingual rendition in host cell. In poliovirus, 2A acts a trans activator of interlingual rendition at IRES when host cell is non imhibited. When 2A pro was mutated it led to loss of cleavage activity in trans but non in Commonwealth of Independent States, and no reproduction of viral RNA was seen therefore this confirms that 2A pro is required for viral RNA reproduction. The aphtho and cardiovirus 2A protease show no sequence similarity to entero/rhino virus although 2A peptidase are similar in size.
The primary cleavage of hepatovirus and echovirus does non take topographic point by the cleavage of 2A protease 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 dwelling of two hydrophobic parts with ? amphipathic a-helix spheres taking to the change of membranes in the septic cells. The virus induced cysts are formed when 2B and its precursor 2BC enter into the host membrane of Golgi and ER complex by changing the permeableness of plasma membrane to organize virporin composite. Once the 2B and the precursor 2BC enters the host there is an instability in Ca2+ homeostatis mechanism and blocks protein conveyance from ER to Golgi and besides initiates anti-apoptosis belongings. The 2B protein besides blocks the activation of IRF-3 in Hepatitis A virus through which the cellular IFN-? cistron written text is inhibited so that there is no injury to Hepatitis A virus in the host. ( Lin et al, 2009 ) . The viral RNA was anchored for the spacial agreement required for reproduction by the 2C protein. The mutagenesis experiments confirmed that the 2C proteins are involved in strand separation of viral RNA while reproduction ( Porter, 1993 )
The secondary cleavage is carried out within the viral proteins and hence it is mediated by 3C protease which plays a really of import function in protein processing and RNA reproduction. The reproduction proteins are generated within the P2 and P3 precursor proteins when 3C ego cleaves at the P3 part of the polyprotein. 3C protease or the 3CD precursors cleaves the poly ( A ) binding protein to suppress viral interlingual rendition during late poliovirus infection. 3C besides cleaves the host cell protein required for written text. The cardinal processing measure for the viral protein processing cascade is initiated by 3C protease. The 3CD proteins besides play a major function in carring out of import maps during RNA reproduction ( Bedard and Semler, 2004 ).
The major map of 3C protease in secondary cleavages is that it process the mirid bug and the replicative protein precursors. The processing of mirid bug in poliovirus is done by 3CD protease and non 3C protease. The 3C proteins are besides used to split assorted figure of host cell proteins such as histone H3, written text factor IIIC, TATA adhering protein and microtubule-associated protein 4. The 3CD protease depends on the host cell protein EF-1 ? and the host cell factor is replaced by 3CD organizing 3AB:3CD protease composite to adhere at the 3 ‘ terminal of the poliovirus genome ( Ryan and Flint, 1997 ) . The 3A protein inhibits the cellular protein map and besides presents the membrane proteins during viral infection. When the poliovirus 3A protein was mutated, the uridlylation of VPg was affected and besides inhibited the viral RNAs plus strand induction ( Porter, 1993 ) .
2B is a viral protein which is required for virion release by changing the cell membrane increasing permeableness which is required for poliovirus RNA reproduction. 2C proteins and its precursor 2BC is necessary for the re-arrangement of intracellular membranes and besides for the viral induced cytoplasmatic cysts. 2C binds with the subtraction strand of poliovirus RNA at the 3 ‘ non coding part and hence it plays a function in positive RNA viral strand synthesis and besides in minus maroon RNA synthesis. 3C and 3D protein helps in immune response intervention and besides in viral RNA reproduction. 3A protein is really much of import because these proteins help the picornaviruses to get away from MHC-I ( Major histo compatability ) look and intracellular membrane conveyance by suppressing both the MHC-I and intracellular membrane conveyance of the host cell. 3B protein besides called as VPg is linked to the 5 ‘ terminal of both the positive and negative strand RNAs. 3AB, 3C, 3CD and 3D are required during the procedure of assembly in reproduction and besides initiates viral RNA polymerase 3D and self cleavage of 3CD. 3C and 3D are involved in adhering viral RNA, protein processing and RNA reproduction. The cloverleaf construction of poliovirus virus and coxsackievirus consist of root cringles in which the viral polymerase precursor, 3CD binds to the root cringle I. 3CD besides binds with the host cell protein poly R ( C ) adhering protein 2 ( PCBP2 ) to assist merely in RNA reproduction. 3D is responsible for VPg uridylylation and RNA concatenation elongation while synthesizing viral RNA because it contains RNA dependant polymerases and even tends for mistake prone and mis-incorporation of 1-2 bases per reproduction ( Bedard and Semler, 2004 ).
VP1, VP2 and VP3 are the three larger mirid bug proteins folded into eight stranded antiparallel ? barrels and a little 4th protein called VP4 is located inside the mirid bug. The 3C peptidase cleaves VP3, VP1 and VP0 at the P1 part. The amino end point of VP0, VP3, VP1 initiates the assembly of the virion atom. VP0 peptides are cleaved into VP2 and VP4 at the concluding phase of processing and assembly. VP4 and VP2 are next to each other at their terminals when cleaved. The infection of the host is initiated when the mirid bug 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 next substrates. Poliovirus receptor molecule is an built-in membrane protein dwelling of one variable and two changeless spheres and this receptor molecule attaches to the host cell to originate reproduction procedure. The general receptors used by the assorted picornaviruses are poliovirus receptor for poliovirus fond regard, 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 canon is formed by flanking the monomers, VP1, VP2 and VP3 which helps the virus to get away the immune response by the host cell. The interaction of mirid bug proteins with the intracellular host factors affects the initiation of programmed cell death ( Lin et al, 2009 ).
Host cell shut off mechanism
The messenger RNA of picornavirus is uncapped and therefore the interlingual rendition takes topographic point by straight presenting ribosomes at internal ribosome entry sites ( IRES ) . The cleavage of eucaryotic interlingual rendition induction factor, eIF4G by 2A peptidase inhibits the cap dependent mRNA interlingual rendition of the host cell. When poliovirus is introduced into the host it inhibits the host cell interlingual rendition taking to apoptotic cell decease. The eIFGII is more immune to the infection of poliovirus than eIFGI. The decease inducement proteins encoded by cellular messenger RNAs were translated by cap independent interlingual rendition taking to apoptotic decease. The cleavage of eIF4GI by caspase 3 besides induces apoptotic cell decease but differs from poliovirus 2A peptidase procedure. Severe suppression of interlingual rendition taking to programmed cell death is seen by both the caspase 3 activity and 2A peptidase. IRES elements in messenger RNA encode proteins which regulate apoptotis. Even poly ( A ) protein and dystropin protein are besides cleaved by 2A protease. Hence, the cleavage of poly ( A ) binding protein by 2A peptidase cause apoptotis and the cleavage of dystrophin protein can bring on apoptotic procedure due to cytoskeleton break ( Goldstaub et al, 1999 ) .
The enterovirus 2B protein suppresses apoptotic tract of the host cell by commanding intracellular Ca2+ homeostatis. The apoptotic responses are initiated by the 3C and 2A protease to suppress cellular written text and cap dependent interlingual rendition. When the 2B proteins are suppressed by caspase-3 activation it leads to apoptotis ( Campanella et al, 2004 ) . The cellular messenger RNA encode for the decease inducement proteins which are translated by cap independent interlingual rendition. When eIF4GI and eIF4GII are cleaved by 2A peptidase it leads to apoptotic decease because it inhibits cap dependent interlingual rendition. The 2A proteins besides 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 constituent of cap adhering composite of cap construction at 5 ‘ terminal of messenger RNA. The 40 S ribosomal fractional monetary unit cheques at the 5 ‘ non coding part until it finds the originating codon or the the reliable start codon AUG and so sends a signal to the 60S to organize a complex. For interlingual rendition to happen in messenger RNA the 5 ‘ non coding part with the cap terminal should adhere with the eIFE and once it binds to the cap the 40 S ribosomal fractional monetary unit scans for the reliable induction or get down codon AUG and one time it finds the AUG codon it gives a signal to 60 S ribosomal fractional monetary unit to organize a complex with 40 S and originate the interlingual rendition along with induction factors ( Bedard and Semler, 2004 ) .
In picornavirus the 5 ‘ terminal of messenger RNA in the non coding part is non capped and therefore to originate interlingual rendition, the cap independent mechanism is required. Hence in picornavirus, the 40 S ribosomal fractional monetary unit scans for ribonucleo protein composite at 5 ‘ non coding part and induction take topographic point to recognize reliable start codon. The eIF4G is cleaved by viral protease 3C and 2A which shuts off the host cell interlingual rendition ( cap dependant ) and besides cleaves poly ( A ) binding protein ( PABP ) and hence it inhibits the host cell interlingual rendition.
When the host cell is infected by Foot and mouth disease virus ( FMDV ) , the eIF4G fractional monetary unit which is responsible for the cap dependent interlingual rendition of the host is cleaved by the L protein and for the Encephalomyocarditis virus ( EMCV ) , the cap dependent interlingual rendition of host is blocked by a represser protein 4E-BP1 which binds at the 5 ‘ cap part and therefore the represser protein 4E-BP1 blocks the binding of eIF-4E which is the cap adhering fractional monetary unit and therefore the host interlingual rendition is shut off. The Hepatitis A virus ( HAV ) does non close off the host interlingual rendition because it requires eIF4G for its interlingual rendition ( Bedard and Semler, 2004 ).
All picornavirus consist of internal ribosome entry site ( IRES ) and are mapped to the 5 ‘ non coding part. There are four types of IRES seen in picornaviruses based on the RNA secondary constructions. 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 interlingual rendition is initiated by two factors
- Canonic induction factors and
- IRES trans-activating factors
Canonic induction factors to originate IRES mediated interlingual rendition:
The IRES elements of poliovirus and EMCV are similar and necessitate the induction factors to be primed at the 40S ribosomal fractional monetary unit. The IRES mediated interlingual rendition is initiated by certain canonical factors such as eIF4G and eIF4B to adhere to the viral RNA and besides certain other fractional monetary units such as eIf3 and eIF2 to pre-bind to the 40 S ribosomal fractional monetary units. The IRES interlingual rendition of poliovirus and EMCV is promoted by the poly ( A ) binding protein ( Lin et al, 2009 ) .
Figure Structural characteristic of IRES elements ( Lin et al, 2009 )
Noncanonical induction factors to originate IRES mediated interlingual rendition:
The polypyrimidine tract-binding protein ( PTB ) is a fifty-seven KDa messenger RNA splicing factor which increases and promotes the activity of IRES in poliovirus. The molecular shift from interlingual rendition to the reproduction of poliovirus is done by the proteolytic cleavage of PTB. The PTB besides functions as RNA chaperon by stabilising the type II IRES of FMDV and EMCV. Lupus autoantigen ( La ) is a fifty-two KDa of atomic protein which binds to certain distinguishable parts of HAV IRES and with little interfering RNA and therefore the HAV IRES interlingual rendition and reproduction is suppressed. Poly ( rC ) binding protein ( PCBP2 ) bind to the type I IRES of picornavirus and hence it leads to internal induction of interlingual rendition in type I IREs elements. The heterogeneous atomic ribonucleoprotein A1 ( hnRNP A1 ) is an RNA binding protein which binds to the 5 ‘ UTR of HRV2 and regulates written text ( Lin et al, 2009 ).
The picornavirus infection has lowered the degree of host cell written text and hence there is a addition in the figure of viral RNA molecules. The atomic localisation signal ( NLS ) nowadays in the poliovirus 3D protein targets the 3CD precursor to the karyon for autocatalytic concluding ripening and let the release of 3C peptidase ( Bedard and Semler, 2004 ) . )
Picornavirus RNA Reproduction
The new positive and negative strand viral RNA are synthesised by utilizing the viral encoded RNA dependent RNA polymerase 3D. The RNA dependant RNA polymerase 3D Acts of the Apostless as a protein primer and signifiers VPg-pU-pU which initiates the viral RNA reproduction and the procedure is known as VPg uridylylation. When VPg uridylylation is done, the poly ( A ) piece of land at the 3 ‘ terminal acts as an induction site for the synthesis of negative strand RNA synthesis. The negative strand developed act as a templet for the synthesis of new viral positive RNA strand by cap independent interlingual rendition. Many or legion transcripts of positive viral RNA strand can be synthesised from a individual negative strand.
In picornaviruses, there are legion RNA sequences and secondary constructions within the 5 ‘ non coding part which are indispensable for the RNA reproduction. The viral protein 3CD and the host protein, PCBP binds at the 5 ‘ cloverleaf construction of the non coding part. The cellular host protein PCBP binds to loop B and the 3CD binds to loop vitamin D of the 5 ‘ cloverleaf construction. A treble composite is formed when 3CD and PCBP2 binds with the cloverleaf construction along with the viral RNA to organize RNA reproduction. When PCBP2 was depleted, there was hapless RNA synthesis in poliovirus and hence it was recognised that PCBP2 play a critical function in RNA reproduction. PABP is a cellular protein that binds to the poly ( A ) piece of land at the 3 ‘ terminal of viral RNA interact with 5 ‘ cloverleaf construction of PCBP2 and the viral protein 3CD. When the PABP at the 3 ‘ terminal interact with PCBP2 at the 5 ‘ terminal, the viral RNA may interact and hence there is an induction of reproduction procedure. Even the 3AB and 3CD proteins interact at the 5 ‘ cloverleaf construction to each other at that place by originating viral RNA reproduction. The cis-acting reproduction component ( cre ) was found within the coding part of picornavirus genomic RNA which is required for viral reproduction and viability. In aphthovirus the cre construction is at the noncoding part of the 5 ‘ terminal. The cre sequence AAACA was found in the conserved hairpin construction at the coding part of picornaviruses required for RNA reproduction. The core elements act as a binding site for viral reproduction proteins and besides as a templet for VPg uridylylation. At cre component, VPg is more efficient than at the poly ( A ) piece of land and uridylylation on cre construction leads to merely positive-strand RNA synthesis and for uridylylation and reproduction induction of the subtraction, strand uses the poly ( A ) piece of land. The interaction between 3CD with cre and cloverleaf RNA may set up the viral RNA structurally for efficient RNA reproduction. The 3 ‘ NCR and the 3 ‘ poly ( A ) piece of land forms the site of reproduction for subtraction strand RNA synthesis. The poly ( A ) piece of land at the 3 ‘ terminal maps for virus viability, impart stableness, and besides for the efficient RNA reproduction by interacting with viral RNA at the 5 ‘ terminal ( Bedard and Semler, 2004 ).
The negative strand is foremost formed by utilizing the VPg protein primer and the VPg plays an of import function in reproduction. 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 ) piece of land templet may besides affect other host proteins for reproduction. The 2C binds at the 3 ‘ end of the negative strand demoing ATPase activity. The RNA polymerase 3D unwinds to develop viral RNA synthesis ( Bedard and Semler, 2004 ).