Proteolytic Activities Involved In The Picornavirus Replication Biology Essay


The picornavirdae family (picorna virus) is known to cause a wider range of well studies diseases, such as hepatitis, poliomyelitis and mycocarditis etc. as compared to other virus families. The illness caused by the viruses may be asymptomatic or cause clinical syndromes. Foot and mouth disease is caused by picornavirus which was the first animal virus to be discovered in 1898 (Acheson, 2007) Poliovirus is the most extensively studied picornavirus for vaccine development. These viruses are 7500nt in length and consist of a 3' poly (A) tail with variable length varying from 65-100nt. This virion RNA has a virus encoded peptide Vpg attached at its 5' terminus. (Racaniello et al., 2006). The term picorna can be split into pico meaning very small (18-30nm) and the RNA referring to the single stranded positive sense RNA that the virus family possesses.


The genome of picornavirus contains an ssRNA (+). The nucleic acid RNA (+) present in picornavirus in considered being infectious. Level of infectivity is elevated if the ssRNA enters into the cell via transfection. 5' UTR consists of clover leaf secondary structure which is called as (IRES) internal ribosome entry site. Both the ends of genome are altered; 5' end by small protein Vpg (covalently attached) and is composed of about 23 amino acids. And the 3' end is modified by polyadenylation. The picorna viruses with the RNA molecules size ranges from 7.2-8.5kb in size lack a lipid envelope. These viruses can be inactivated by ionizing radiation, phenol and formaldehyde. After infection translation is initiated with help of ribosomes which leads to synthesis of polyprotein molecule (weight 250, 000)

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Picornaviruses have been classified according to Baltimore Classification system of viruses. The picorna virus consists of 12 genera

1. Enterovirus 2.Cardioviruses 3.Hepatoviruses

4. Rhinovirus 5.Aphthoviruses 6. Parechovirus

7. Erbovirus 8. Kobuvirus 9.Teschovirus

10. Sapelovirus 11. Senecavirus 12. Tremovirus

Rhinoviruses mainly infect throat and nose unlike enteroviruses which mainly infect enteric tract. Both the genera have identical morphology but they can be identified based on clinical, biophysical and epidemiological studies.

Table1: Genre in Picivirdae family (Ref:

The nuclecapsid of a picorna virus has a twelve penton structure, one at each vertex of the virus. Surrounding each penton there are five other proteins. The virus has a protein complex which resembles a triskelion structure on its each face. This is in turn in contact with six other proteins. The region between the pentons and triskelion is called a canyon, to with the cell surface binds in and conformational changes takes place to the viral proteins.

The capsid of these viruses consists of 60 promoters densely packed in an icosahedral arrangement. Picornavirus has a nonenveloped structure. The 3-D structure of picornavirus was determined using X-Ray crystallography. Each of the 60 promoters consists of 4 polypeptides, Etoposide (VP) 1, 2, 3 and 4. These polypeptides are derived from the cleavage of a larger protein. The main functions of the capsid proteins are to protect the virus from environmental RNAses, host and cell tropism, delivering the viral RNA to the cell cytoplasm by penetrating the host cell and selection and packaging of viral RNA. VP1, VP2 and VP3 wrap themselves into a jelly shape roll or wedge shapes structure which is composed of β barrel 8 stranded structures. Capsid of picornavirus is formed by these 3 proteins unlike VP4 which is present below the shell. The capsid proteins of different plant and animal viruses share similar folds.

Many picornaviruses contain depressions called as pits or canyons on the surface of capsid that plays role in attachment to the surface of receptors. Depression or canyon or pit is not found in other types of picornaviruses. Foot and mouth disease virus and rhino virus make use of enodcytosis (receptor - mediated) to enter the cell. Acidification caused in the endosomal vesicle leads to conformational change within the capsid causing breakdown of subunits of capsids and viral RNA release. In other types of picornaviruses such as poliovirus experience conformational alteration on the cell surface directly on attaching with the receptor.

Figure 1: shows the general replication pattern of Picornaviruses

(Ahlquist et al., 2003).

The duration of a replication in picornavirus is 8-10 hours. The cycle duration may vary depending on pH, temperature, cell types and number of viral particles that infect the cell. The general process of replication is that the virus comes in contact with the host cell. Enters the cell by means of endocytosis and the uncoating process happens. Then the viral RNA are transcribed and translated by cell enzymes and ribosomes. The cells productivity is taken over by the virus and new virus particles are produced by the cell. Within 30 minutes of the virus infection, the cellular protein synthesis declines almost reaching zero. This is called 'SHUT OFF'. The SHUT OFF is due to the cleavage of the cellular protein eIF-4G, a component of 220KD cap-binding complex (CBC or CBP).

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The picorna viruses attach themselves to the host cell by specific receptors. In the case of polio virus, a type of picorna virus the receptor protein is CD155 whose function is that it binds with the protein called vitronectin, which is an adhesion protein. Similarly most of the rhinoviruses bind to receptor which is an adhesion protein called CD54 or ICAM-1 (Intracellular adhesion molecule-1). Lots of information regarding picornavirus replication came into picture after conduction of single-step growth curve experiments. Replication either occurs in cytoplasm or in enucleated cells. The replication in this case cannot be inhibited by the action of actinomycin D. RNA dependant RNA polymerase produces (-) strands copying the genomic RNA A replicative intermediate complex which is multi stranded help in RNA synthesis. (-) RNA acts as a template for (+) strands. Two possible replicative models have been suggested after extensive transcription studies (in vitro).The total number of (+) strands synthesised exceeds the number of (-) strands by ratio of 40:1. During first viral infection RNA of virus is formed in combination with membrane vesicles. These vesicles play role in localizing the elements needed for sufficient RNA replication in the host cell. Picornavirus proteins 2C, 2B, and 3A help in inhibition of protein secretion as well as inhibition of membrane trafficking via golgi apparatus to plasma membrane. This leads to formation of vesicles in the infected cell.

Fig2: Replication mechanism of Picornavirus (Microbiology Bytes, 2007).

The interaction between capsid and genome although is not very clear but still RNA is considered to be packaged into the pre formed capsids.The capsid is formed after the cleavage of precursor of P1 polyprotein into promoter comprising of VP3, VP1 and VPO. They combine together thus enclosing the genome. Viral genome is packaged in the capsid in the presence of substances such sodium to cancel (-) charge caused by phosphate group on RNA. Diameter of viral particle is 27-30 nm depending on dehydration degree.

Fig3: Diagram showing the cleavage of polyprotein to form mature virion


For instance when we consider the binding of Rhinoviruses to the cell via ICAM-1. The RNA enters through the membrane via the centre of the penton after the viral protein had embedded to the cell membrane. The VP1 molecule from the central part of the penton surrounds a closed pore. The canyon will be present where the VP1 Opposes VP2/3. At the floor of the canyon, there is a narrow hole bellow which a bigger space called pocket is present. The pocket contains 'pocket factor'. Another protein VP4 is present below VP1, 2 and 3 which cannot be seen from outside. The ICAM-1 fits into the VP1/2/3 complex, which fits into the canyon but this does not penetrate into the pocket. When ICAM binds in the canyon, a conformational change which tips back VP1 from centre of the penton takes place. This squeezes the pocket and possibly the binding displaces the pocket factor. When VP1 tips back, the pore opens as VP3 moves out of the way. This allows VP4 to move through the pore. The formation of a pore is also helped by the fact that the VP1 amino terminus is an amphipathic alpha helix which flips out ICAM-1 binding.

Fig4: Diagram to show translation process in Picornavirus


After the virus binds to the protein and enters the cell the process of uncoating takes place. There is a possibility that the virus after binding might get eluted again. When that happens the virus complex go through the first step of uncoating by losing the VP4 protein with the exclusion of non infectious A particle. And when the virion binds to the membrane of the host, it forms a coated pit structure and undergoes the above reactions and finally the RNA from the shell is released into the host cytoplasm and empty shells are expelled out.

The host cellular functions are influenced by viral proteolytic activities. The major proteases involved are the 2A and 3A which includes Lpro, 2Apro, and 3Cpro. These are picorna virus encoded proteases which are important for viral polyprotein processing (Strebel et al., 1986, Joyoda et al., 1986). The viral proteases not only cleave viral polypeptides but also inhibit various host mechanisms. The 3Cpro enters the nuclei through its precursor 3CD' or 3CD, which has a nuclear localization sequence (NLS) (Sharma et al., 2004). The 3Cpro cleaves numerous factors and regulators associated with cellular DNA-dependent RNA polymerase I,II and III such as TATA box binding protein (TBP), Octomers-binding proteins (OCT-1), transcription activator p53, cyclic AMP-responsive element binding protein (CREB), histone H3 and DNA polymerase III (Weidman et al., 2001). 3Cpro may also be involved in virus-induced blockage of host transcription. 2Apro cleaves TBP but cannot inhibit cellular transcription (Yalamanchili et al., 1997). Numerous cytoskeleton-associated factors are cleaved by 3Cpro and 2Apro.

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The 2Apro of enterovirus and rhinoviruses carryout the primary cleavage between the capsid and non-structural precursors on the respective polyproteins. The enzyme cleaves at the C terminus of VP1 and its own N-terminus. The Lpro of FMDV in contrast cleaves between its own C-terminus and N-terminus of VP4. Both the 2Apro and Lpro are responsible for specific proteolysis of a host cell protein, the eukaryotic initiation factor (eIF) 4G (Lloyd et al., 1988). The cleavage of eIF4G inhibits the cell to initiate the protein synthesis on its own mRNA. The viral protein synthesis is initiated internally by the ribosomes on the IRES (Internal ribosome entry segment) within 5'UTR (Jackson et al., 1990). The eIF4G cleavage does not occur at the same peptide bond. Lpro cleaves between residues Gly479 - Arg480 and 2Apro cleaves at Arg486 - Gly487. Though the cleavage takes place at different bonds, the effect of the cleavage is same.

A study by Ryan et al., (1997), shows that 3Cpro is unlike the other two proteases. The 3Cpro is responsible for a series of secondary cleavages resulting in the processing of capsid and replicative proteins precursors. The 3Cpro mediated cleavages occur at Gln-Gly pairs. The pairs within the protease are not processed. The processing of the capsid protein precursors is actually mediated by 3CDpro, not by 3Cpro. The autoprocessing of the 3CDpro to 3C and 3D is enhanced by pure 3AP protein addition. This also stimulates the processing of proteins 2BC.The ability of a poliovirus 3Cpro to process precursors was studied by constructing chimeric polyproteins in which the regions of the poliovirus cDNA encoding 3C was replaced by equivalent sequences from human rhinovirus 14 (HRV 14). The poliovirus P2 precursors could be processed by both the HRV-14 and CB3 3Cpros, but the P1 capsid protein precursors was not processed by either HPV-14 or CB3 3Cpros.

The membrane alterations in the infected cells are caused by viral protein 2B and its precursor 2C. The 2B and 2BC contain two hydrophobic regions, which are α helix domains. This helps the protein to integrate into the membrane of the host golgi and ER complex which produces virus induced vesicles and virporin complex. The accumulation of 2B or 2BC protein leads to plasma membrane and disassembly of gogi complex.

The 3A protein is a membrane binding protein which inhibits cellular protein secretion and mediating presentation of membrane proteins during viral infection. The 3A protein expression causes ER to Golgi trafficking. The protein trafficking by 3A is caused by the disruption of ADP-ribosylation factor (Arf) family, which is an important component of the membrane secretion pathway (Belov et al., 1997).

The 3AB protein is a multifunctional protein with the 3A portion of the protein associated with membrane vesicles (Jowner et al, 1996). This interaction anchors the replication complex to virus induced vesicles. The membrane associated 3AB protein binds directly to precursor 3CD on the RNA of poliovirus. This stimulates the activity of the 3CD and serves as an anchor for 3D polymerase in the RNA replication complex. The 3AB functions as a substrate for 3D polymerase in VPg uridylylation. The 3AB of the poliovirus was observed to show other functions such as helix stabilization. This shows that 3AB has nucleic acid chaperon activity in destabilizing the secondary structure of RNA and enhances the hybridization in complimentary nucleic acids in viral replication (Destefano et al. 1999).

The enteroviral and rhinoviral 3B proteins (VPg) are small peptides. This protein consists of 21-23 amion acids, which are covalently linked with the 5'tyrosyluridine in the tyrosin residue in VPg. The uridylylated VPg is utilized as a primer in both positive and negative RNA synthesis.

The 3CD protein which is a precursor of mature 3C protease activity but no polymerase activity. The 3CD helps in viral RNA replication by circularization of the viral genome via interacting with both 5' and 3' ends of viral RNA (Harris et al., 1992). PCBP1 and PCBP2 are KH domains RNA binding proteins, which are involved in the metabolism of cellular mRNA's in normal cells. The addition of recombinant PCBP1 rescues viral RNA replication in PCB depleted extracts but does not rescue viral translation (Harris et al., 1992)

The 3D protein is one of the major components of the viral RNA replication complex. It is a RNA-dependent RNA polymerase. Elongation activity is observed in the purified poliovirus 3D polymerase (Van Dyke., 1980). 3D polymerases can also uridylylate VPg and use VPg-pUpU as a primer during viral RNA replication (Paul et al., 1998).The polymerase-polymerase reaction of poliovirus has been observed in biochemical and crystal structure studies. The polymerase oligomerization has been proposed to be responsible for efficient template utilization. The RNA secondary structure in the viral genome plays 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 circularization of the polioviruses template between the 5' and 3' terminal of the viral genome is crucial during the initiation of both positive and negative strand RNA replication (Pata et al., 1995). 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 3' stem-loop I of the negative strand is the initiation site of positive - strand RNA synthesis. Some cellular proteins such as La can interact with both 3' and 5' UTR's 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 (Pata et al., 1995).

Fig 5: Shows the summary of the Proteolytic activities taking place in the replication of Picornavirus

(Journal of biomedical science, 2009).

The proteolytic activities taking place inside the picorna virus is responsible for its replication. When a host is infected by the picorna virus the proteolytic activity of the host gradually reaches zero. This is called SHUT OFF and this is caused dude to the proteins synthesised by the picornavirus. The main proteases are the 2Apro, Lpro and 3C pro. The breakage of proteins leads to the process of replication of these viruses. The proteolytic activity of the host is disrupted by the cleavage of eIF-4G. The picornavirus will not replicate if any one of these proteases are not synthesised. Thus the modern drugs to cure diseases caused by picornoviruses target these protein production pathways.