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Picornaviruses are a large family of animal viruses which are pervasive in nature. This belongs to the family picornaviridae and consists of 12 geners they are Enterovirus, cardiovirus, aphthovirus, hepatovirus, parechovirus, Erbovirus, Kobuvirus, Teschovirus, Sapelovirus, Senecavirus, Tremovirus and Avihepatovirus (http:/www.picornaviridae.com/). These viruses are very small, non-segmented, single-stranded, positive sense RNA viruses. The virion is naked 30-nm icosahedrons. The capsids are composed of 60 copies of four nonidentical virion polypeptide chains (VP1, VP2, VP3, and VP4). The RNA codes for only one long open reading frame. Translation proceeds primarily from a single strong initiation site and produces a giant precursor polyprotein (Mr = 250,000) that is divided into three regions, they are 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). Two groups of picornoviruses are of medical importance, the enteroviruses that parasitise the entire tract and the rhinovirus that infect the nasal mucosa. (Ananthanarayan, Paniker, 2005) Picornaviruses are acid-resistant, which enables them to survive passage through the stomach. They replicate in the small intestine, can readily be isolated from faeces and are spread by the faecal-oral route. Gastroenteritis is, however, not a major feature of Picornaviruses infections (Coller.L, Oxford.J, 2006).
Each member of the family picornaviridae expresses its viral gene products through the IRES-directed translation of single ORF that encodes approximately 250-Kda polyorotein (figure 1). The arrangement of viral proteins in the picornavirus polyprotein is hichly conserved between members of the nine genera, with structural proteins that is P1 or capsid found in the amino-terminal portion of the polypeptide, followed by non structural proteins that is P2 and P3 proteins, which interact with the viral RNA, and other non structural proteins and various host cell components during the viral life cycle. Five picornavirus genera have a polypeptide, termed L or leader protein, at the amino terminus of their polyprotein immediately preceding the P1 (capsid) region; although not present in enteroviruses, rhinoviruses, hepatoviruses or parechoviruses, this protein is included in the summary diagram (Figure 1) (Whitton et al, 2005).
Figure 1: A diagrammatic representation of the picornavirus genome is shown, combining features from all of the genera. The 12 mature polypeptides are shown, together with the three main cleavage intermediates. The main biological functions are included for each polypeptide. IRES, internal ribosome entry site; UTR, untranslated region; VPg, viral protein genome-linked (Whitton et al, 2005).
The viral polyprotein is processed both during and after translation with exquisite specifically, at amino-acid sequences recognized by proteases encoded in the viral genome. This process was initiated by one or more primary cleavage events carried out in cis, followed by a series of trans cleavages that generate the mature polypeptides. The intermediate cleavage products have different functional specificities from their mature counter parts. Most of the cleavages events along the viral polyprotein occur between Gln-Gly pairs and are mediated by the 3C polypeptide, a chymotrypsin-like protease. At least two other proteolytic activities are encoded by Picornaviruses. For most picornavirusis, the 2A protein is a chymotrypsin-like protease that has a role in polyprotein cleavage, and it might also be important in viral RNA replication. Picornaviral genome replication takes place in a viral replication complex (figure 2) and depends on the RNA-dependent RNA polymerase 3D, which is the most highly conserved polypeptide among members of the family Picornaviridae (Whitton et al, 2005).
HOST FACTORS AND CAPSID PROTEINS INVOLVED IN RECEPTOR BINDING
Picornaviruses contain capsid proteins that are encoded by the P1 region of the genome, and these capsid particles comprise 60 copies of 4 P1-encoded poly peptides, they are VP1, VP2, VP3, VP4. The first three viral proteins (VP1-VP3) reside on the outer side of the virus, and the VP4 is located completely in the inner surface of the capsids. The capsid proteins mediate the initiation of infection by binding to the receptor on the host membrane. Many picornavirues have similar receptor molecules that are from immunoglobulin super family (IgSF), whose extracellular regions comprise two to five amino-terminal immunoglobulin like domains. In this receptor the amino terminal domain, D1 is involved in bonding of aminoacid residues of the picornavirus canyon, which can trigger viral instability and uncoating. The receptor recognition is important in derermining the tropism of the cell and host range. However, the interaction of capsid proteins with intracellular host factors is also significant. Factors other than viral-receptor interaction, including cellular factors and viral genome elements, both interact with 5' untranslated region and thus influence the effiency of translation, initiation and virus replication (Hughes J. P, Stanway G, 2000).
PICORNAVIRUS PROTEOLYTIC ACTIVITIES INFLUENCE CELLULAR FUNCTIONS
The picornaviruses encode all of their proteins. The translation products are not fully observed, this is due to extremely rapid co-translational, intramolecular (in cis), or primary cleavages. Subsequent secondary processing of the primary cleavage products may occur either in cis or in an intermolecular fashion (in trans). Such processing of the replicative protein precursorforms (P2 and P3) may be regulated to follow alternative, mutually exclusive, pathways to generate different sub-sets of biochemical functions from the same type of precursor molecule. The genera within this family show differences in their genome organization, particularly in relation to their proteolytic activities. Picornavirus polyproteins contain three recognizable types of proteinase the Leader, 2A and 3C proteases (Lpro , 2Apro ,3Cpro) are picornavirus-encoded proteases, viral proteases not only cleave viral polypeptides, but also inhibit various host mechanisms. Picornaviral 3Cpro can enter nuclei through its precursor 3CD1 or 3CD, which contains nnuclear localization sequence (NLS). 3Cpro can cleave numerous factors and regulators that are associated with cellular DNA-dependant RNA polymerase I, II, III, such as TATA-BOX binding protein (TBP), octamer-binding protein (OCT-1), transcription activator P53, cyclic AMP-responsable element binding protein (CREB), histone H3 and DNA polymeraseIII. 3Cpro may be involved in virus-induced blockage of host transcription. 2Apro reportedly cleaves TBp, but cannot inhibit cellular transcription.
2Apro and Lpro cleave eIF4GI and eIF4GII, 3Cpro cleaves eIF4AI, which lead to the shut off of host translation. The cleavages of Poly (A) binding protein (PABP) by 2Apro and 3Cpro also contributes to the inhibition of cellular translation. Furthermore, EMCV 2A protein without a catalytic function reportedly associates with 40S ribosome subunit, suggesting another mechanism of host translation shut off. The components of nuclear pore complex were degraded in infected cells and 2Apro is the factor that blocks nucleo-cytoplasmic trafficking (Ryan D.M, Flint.M, 1997).
The aphthoviruses, together with equine rhinoviruses (ERV) 1 and 2, possess a proteinase (Lpro) at the N terminus of the polyprotein not found in other genera. Lpro cleaves cotranslationally at its own C terminus and exists in at least two formsthey are Labpro and Lpro derived from initiation of translation at either of two in-frame AUG codons located 84 nucleotides apart.The Lbpro form undergoes a post-translational modifcation by a carboxypeptidase B-like activity producing Lb' Both the Labpro and Lbpro forms are able to cleave at the L/P1 junction either in cis or in trans (Ryan D.M, Flint.M, 1997).
The entero and rhinoviruses, a primary cleavage occurs between the P1 capsid protein precursor and the replicative domains of the polyprotein at a tyrosine-glycine scissile pair. This cleavage is mediated by a virus-encoded proteinase, cleaving at its own N terminus. In addition, a processing intermediate from the P3 precursor of both rhino and enteroviruses may be processed by 2Apro at a tyrosine-glycine scissile pair within 3D to yield not the 3C proteinase and polymerase but 3C' and 3D' (Ryan D.M, Flint.M, 1997).
PICORNAVIRUS 3C PROTEINASE
All picornaviruses possess a 3C proteinase (3Cpro) which shows a high degree of sequence similarity across the genera. Like Lpro and 2Apro, 3Cpro mediates a single primary cleavage, although at a distal site between 2C and 3A. Unlike other picornavirus proteinases, 3Cpro is responsible for a series of secondary cleavages resulting in the processing of the capsid and replicative protein precursors. 3Cpro from other genera is, however, able to process capsid protein precursors although the processing may be more efficient with 3CDpro. 3D is an RNAdependent RNA polymerase but in this context the presence of 3D serves to alter the substrate specificity of 3Cpro in the processing of capsid protein precursors in trans.3C also retains proteolytic activity in the precursor forms 3ABC and P3. The picornavirus 3Cpro has been demonstrated to cleave a number of host-cell proteins, histone H3, transcription factor IIIC, TATAbinding protein and microtubule-associated protein 4 (Ryan D.M, Flint.M, 1997).
PICORNAVIRUSES VIRAL PROTEIN INVOLVED IN CLEAVAGES
3C, 3CD, and 3D: The 3C regions of all Picornaviruses code for a protease with a critical function as the enzyme responsible for majority of maturation cleavages in the precursor poly protein. Proteolysis by 3Cpro occurs in a complex and incompletely understood cascade of cis and trans cleaves at mainly Q-G, Q-S, Q-A, and Q-N pairs. The 3Cpro also induces the specific cleavage of histone H3, because the deleted part of the histone H3 in amino-terminal region corresponding to the presumes regulatory domain of transcriptionally active chromatin, this specific cleavage is related to the severe inhibition of host cell. 3Cpro is sensitive to thiol-blocking reagents, the 3Cpro is structurally and functionally releated to the large subclasses of trypsin-like serine proteases, with a catalytic triad of His-40, Asp/Glu-71, and Cys-147. The 3D regions of all Picornaviruses code for an RNA-dependent RNA polymerase which has regions of homology with all known DNA and RNA polymerases. The recombinant 3Dpol of Picornaviruses is capable of copying plus strands in vitro provided that an oligo (U) primer is present, but one or another of the host factors can circumvent the need for such a primer. The 3Dpol completed the synthesis of a minus strand and provides signal for assembly of the RNP complex on the 5' end of the neighboring plus strand. The complex catalyzes the formation of VPg-PU (-PU) via 3AB. And the complex then catalysis the initiation of the new plus strand in the trans by VPG-PU(-PU) at the 3' end of the neighbouring minus strand newly exposed by the formation of complex. The source of 3Dpol for plus-strand synthesis is present in the RNP complex (Porter G.A, 1993).
VIRAL AND HOST PROTEINS INVOLVED IN RNA REPLICATION
Picornavirus causes a change in host membrane permeability and the production of membranous structures, on which viral replication depends. This viral replication complex has been identified to associate with virus-induced membranous vesicle. There are various replication-associated viral proteins, such as 2B, 2BC, and 3A amd 3D and host proteins (Table 1).
Viral protein 2B is precursor and 2BC is responsible for membranous alteration in infected cells. The cellular proteins of COPII have been reportedly been used in viral-induced production of vesicles. This 2B and 2BC contain hydrophobic regions, they are α-amphipathic, a-helix domain, and these forms virporin complex. The accumulation of 2B and 2BC proteins on Golgi changes the permeability of plasma membrane, and the dis assembly of Golgi complex, and causing cell lysis. The disruption of Ca2+ homeostasis by 2B/2BC is the mechanism why the transport of protein from ER to Golgi is blocked. The 2B-induced intracellular Ca2+ imbalance is also related to the anti-apoptosis property (Yi Lin et al, 2009).
This 3A protein is an membrane binding protein, plays a role in inhibiting cellular protein secretion and mediating presentation of membrane proteins during viral proteins, 3A and 3CD, an recruit ADP-ribosylation factors ( ArFs) to bind to membranes by different mechanisms. The expression of 3A results in recruitment of ArFs to membranes by specifically recruiting the cellular guanine nucleotide exchange factor (Yi Lin et al, 2009).
The 3AB protein is a multifunctional protein. The hydrophobic domain in the 3A protein of the protein associates with membrane vesicles. The 3AB has been demonstrated to function as a substrate for 3D polymerase in VPg uridylylation. The 3AB protein, rather than 3B has been proposed to be delivered to the replication complexes for uridylylation.The 3B proteins contains 21 to 23 amino acids, which are covalently linked with 5' termini of Picornavirus genome via a 5' tyrosyluridine bond in the conserved tyrosine residue in the VPg. The udidylylated VPg is utilized as a primer in both positive and negative-strand RNA synthesis (Yi Lin et al, 2009).
The 3CD protein is the precursor of mature 3C protease and 3D polymerase activity. PCBPs contain four is forms in mammalian cells, PCB1 and 2 are KH domains RNA binding proteins, which are involved in the metabolism of cellular mRNAs in normal cells. Another cellular protein, heterogeneous nuclear ribonucleoprotein K (hnRNP K), interacts with stem loops I-II and IV of EV71 5' UTR. During EV71 infection, hnNP K was enriched in the cytoplasm where virus replication occurs, whereas hnRNP K was localized in the nucleus in mock-infected cells. viral yields were found to be significantly lower in hnRNP K. Knockdown cells and viral RNA synthesis was delayed in hnRNP K knockdown cells in comparison with negative control cells treated with small interfering RNA. Moreover, 3CD has been shown using the pull-down assay, to interact with heterogeneous nuclear ribonucleo protein C (hnRNP C). The hnRNP C participates in pre-mRNA processing in normal cells. The mutant form of hnRNP C with the defective. Theactivity in protein-protein interaction inhibits the synthesis of viral positive-strand RNA, implying the participation of hnRNP C in RNA replication. The interactions of 3CD with these cellular proteins, PCBP, hnRNP C and the viral protein. The transcription factor OCT-! And the nucleolar chaperone B23, have also been identified as co-localizing in nuclei with HRV-16 3CD during virus infection. The HRV-16 3C from the precursor 3CD may play a role in shutting off host cell transcription in nuclei. And also exhibiting protease activity (Yi Lin et al, 2009).
The viral RNA-dependent RNA polymerase 3D is one of the major components of viral RNA replication complex. 3D polymerase can also uridylylate VPg and use VPg-pUpU as a primer during viral RNA replication. The polymerase oligomerization is responsible for template utilization. Sam 68, an RNA binding protein, mediates alternative splicing in cells in response to an extra cellular signal (Yi Lin et al, 2009).
Table 1: Cellular proteins involved in picornaviral RNA replication
Binding sites/associated with viral proteins
Cloverleaf, 3CD, 3C
Negative strand 3'stem-loop I, 3D, 3CD, P2, P3
3CD, stem-loop I and poly(A) tail
3' and 5' UTR
(Yi Lin et al, 2009).
CIS ELEMENTS INVOLVED IN RNA REPLICATION
The RNA secondary structures in viral genome play important roles in the replication of viral RNA. The cis elements contain stem- loop-I at the 5' terminus of 5' UTR, 3' UTR and poly(A) tail at the 3' terminus of enterovirus RNA. The 3CD and PCBP3 on stem loop I at the 5' terminus of 5' UTR and poly(A)- binding protein (PABP) and 3CD on the 3' termini of genome are involved in the circularization of RNA genome for initiation of negative-strand RNA synthesis. The 3CD or 3D has the ability for the interaction with the 3' UTR element. The binding of 3CD and 3AB to 3' UTR does not depend on the interaction with host proteins and suffices for viral RNA replication. The immunodepletion of nucleolin from cell free extract reduced virus reproduction, indicating that nucleolin may be involved in viral RNA replication.
The 3'stem-loop I of the negative-strand RNA is the initiation site of positive-strand RNA synthesis. Positive-strand RNA synthesis is initiated by the recruitment of uridylylated VPg containing replication complexes close to the 3' stemloop I of the negative strand. Viral protein 2C has been reported to interact directly with the 3' stem-loop I of the negative strand. Some cellular proteins, such as La, can interact with both 3' and 5' UTRs of CVB3 independently of the poly(A) tail, and may play a role in mediating cross-talk between the 5' and 3' ends of CVB3 RNA for viral RNA replication. Synthesis of the uridylylated VPg is the first step of viral RNA synthesis. The efficient uridylylation of VPg requires 3D polymerase, 3CD protein, UTP and the cre motif from the viral genome as the template. 3CD has been shown to stimulate cre-mediated VPg uridylylation. Moreover, the 3AB protein is regarded as the precursor of VPg (3B) in the RNA replication complex for VPg uridylylation (Yi Lin et al, 2009).
SWITCH FROM TRANSLATION TO RNA REPLICATION
The positive-stranded RNA viruses use the same RNA as a template for translation and replication. Ribosomes move from the 5' end to the 3' end of RNA to undergo translation, and RNA polymerase binds to the 3' end of the same RNA to initiate replication. While using puromycin to release the ribosomes allows facilitates RNA replication. These two events cannot occur at the same time, so the balance between translation and replication is important (Yi Lin et al, 2009).
PICORNAVIRUS PROTEASES FOR PROCESSING AND AFFECTING NORMAL CELL PROCESSES
The life cycle of picornaviruses is summarized in figure 2, and the genome structure and organization are presented in figure 1. The entry into the cell, the virus undergoes uncoating, and the viral genome is released. All picornavirus genomes are positive-sense RNA molecules encoding a single, long open reading frame (ORF) flanked by lengthy untranslated regions (UTRs), and contains a second short ORF. Formation of this nucleoprotein complex is an absolute requirement for viral RNA replication. Protein-protein interactions between 3CD, PCBP and the host protein PABP (poly(A)-binding protein) have been described, indicating that the cloverleaf-3CD-PCBP that is bound to the viral poly(A) tract. A second important feature of the 5'-UTR is the internal ribosome entry site (IRES), which acts as a 'landing pad' for ribosomes. The 5' ends of most cellular mRNAs have a 7-methyl guanosine (m7G) cap structure, which is recognized by the eukaryotic eIF-4F cap-binding complex as an early event in the translation of cellular proteins. This cap is absent from the 5' end of picornavirus RNAs, which therefore cannot undergo cap-dependent translation, and translational initiation in these viruses instead depends on the IRES. This strategy might have originally developed within the eukaryotic cell and subsequently been appropriated by viruses, because some host-cell mRNAs contain IRESs and undergo cap-independent translation (Whitton et al, 2005).
Figure 2: Picornavirus affecting the normal cell
(Whitton et al, 2005).
EFFECT OF PICORNAVIRUS PROTEOLYSIS OF eIF4G AND PABP IN HOST CELL
In the absence of eIF4F complex, the + ve stranded viral messenger RNA is translated by an internal ribosome entry mechanism. In the 176-kD protein, the eIF4G component was cleaved by the protease 2A which is encoded by the virus. Binding site for in ther eIF4E, PABP located in the NH2-terminal cleavage product while for eIF3 and eIF4A in the COOH-terminal (Figure 3). It was hypothesized that for recruiting ribosome to the 5' end of host mRna cell,there was enough eIF4G fragment in the N-terminal. Viral mRNA translation was shown to be mediated by an internal ribosome entry site (IRES), an approximately 500 nucleotide RNA sequence in the 5' noncoding region of the viral RNA, which was postulated to recruit ribosomes without the need for an intact eIF4F. However, it was noted that cleavage of eIF4G precedes inhibition of host protein synthesis, and conditions could be found in which eIF4G was efficiently cleaved in infected cells in the absence of host translational shutoff. Hence for protein synthesis shutoff, there was no proper cleavage of eIF4G (Bushell.M, Sarnow.p, 2002).
Figure 3: Alterations of the cap binding protein complex eIF4F in infected cells. (Top) Cleavage of eIF4G by picornaviral proteases. (Middle) Sequestration of eIF4E by 4E binding proteins (4E-BP) due to dephosphorylation of 4E-BPs in picornavirus-infected cells. (Bottom) Eviction of PABP by viral NSP3 from the cap binding protein complex eIF4F in rotavirus- infected cells. Interactions of eukaryotic initiation factors eIF4G (4G), eIF4A (4A), eIF4E (4E), and eIF3 (3) are indicated (Bushell.M, Sarnow.p, 2002).
EFFECT OF PICORNAVIRAL PROTEINS ON THE HOST CELL
Many picornavirus proteins exere powerful effects on the host cell, most of the picornavirus infections is the rapid and profound shutdown of host translation, achieved by cleavage of the cellular protein eIF-4G, a key factor in cap-dependent translation. The virus does not disable its prey, it exploits the remains. The eIF-4G cleavage binds preferentially to viral RNA and facilitates IRES dependent translation of viral polyprotein. The actions of this virus can use the eukaryotic translational apparatus exclusively for its own gene expression. The protein enters the nucleus and associates with nucleoli and altering newly formed ribosomes and causing them to preferentially translate IRES-driven messages. Host-cell transcription is also dramatically reduced by several picornavirus infections. Transcription by all three types of eukaryotic RNA polymerase is terminated, creating a disproportionately higher number of viral RNA molecules in the host cell. This effect seems to be mediated by the 3C protease, which cleaves several cellular transcription factors between Gln-Gly pairs. But the life cycle of picornaviruses is cytoplasmic, and the 3C protease contains no obvious nuclear localization signal (NLS).Picornaviral proteins also affect cellular processes other than translation and transcription. The CVB 2A protein is thought to cleave dystrophin, thereby possibly contributing to DCM (Whitton et al, 2005).
Picornaviruses induce inhibition of host-cell cap-dependent protein synthesis. This was mainly achieved through cleavage of eIF4G and/or dephosphorylation of 4E-BP1. The infection was tested on host-cell protein synthesis in order to know, whether they also induce host shut-off. Infection of cells leads to inhibition of cellular protein synthesis. This is accompanied by the cleavage of the eukaryotic translation initiation factors eIF4GI and eIF4GII in a manner reminiscent of that induced by picornaviruses. However cleavages occurs in many sites.(willcocks et al, 2004).
Infection by a Picornavirus leads to a number of effects on the functions of macromolecules in the host cells. They include shutoff of cap-dependent translation, shutoff of host cell RNA synthesis, induction of cytoplasmic vesicles and alternation on intracellular transport pathways between the endoplasmic reticulum and Golgi apparatus. The viral protein 2A encodes for Enterovirus and Rhinoviruses and viral protein L encodes for Aphthoviruses. These proteinases are encoded by Picornavirus and helps cleave eIF-4G. This eIF-4G is one of the essential component required for the recognisation of 5' end of capped cellular mRNAs. Thus the infected cells are unable to transfer the cellular mRNAs and allows the translation of cap independent viral RNAs to take over the cellular protein synthesis machinery. The mechanism and the replication of Picornavirus in the host cells controls the metabolism of the host cells and inhibits their action in all possible ways. The virus protect themselves against the immune response. But in such emphatic action might carry with it a signal disadvantage, viral replication takes several hours and, if the host cell were to die in the meantime, the virus might jeopardize its own propagation. The Picornaviruses trigger the apoptotic pathway and that, but before cell death has ensued, they terminate the process and initiate the alternative pathway. The virus might prolong the agony of the infected cell, furnishing itself with the requisite time to complete its replication. Thus the drugs are designed to target the proteinases that are being synthesized by the virus during the process of their replication. The drugs now act by controlling the growth of the Picornavirus by inhibiting the action of the proteinases of the virion.