Roles Of Proteolytic Activities In Picornavirus Replication Biology Essay

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Viruses are everywhere around us, comprising of enormous proportion of our environment both in number and total mass. Viruses consist of a nucleic acid genome that is either a DNA or RNA and the genome is enclosed by a capsid made up of proteins. Viruses are infectious and they are called as Virions. They multiply inside the cells by expressing and replication the genome and hence they cause disease to the host cells. The viruses can be detected and measured with the Plague assay, Hemagglutination and counted using the Electron Microscope. This Picornavirus is a virion that posses a naked isohedral capsid with a positive sense linear ssRNA as its genome. This virus is of great importance as they cause paralysis, myocarditis, hepatitis, meningitis and common cold in humans. Also they cause foot and mouth disease in cattle (Acheson, 2007). Thus there is a need to understand the mechanism or replication of the virion and also understand the functions of the proteins encoded by these virus particles so that the remedial measures can be formulated for the infection. Also they help to study the physical characteristics of other viral species. The distinctive characteristics of Picornavirus are that they have their translation process being initiated at the Internal Ribosome Entry Site (IRES). The viral proteinases help in the self cleavage of the polypeptide and the genetic material replication is associated with the vesicles in the cytoplasm (Madigan, et al, 1997).

Picornavirus is a virus belonging to the family picornaviridae. They are non enveloped, positive stranded RNA viruses with an icosahedral capsid. The genome RNA is unusual as it has a protein in its 5' end that is used as a primer for transcription by RNA polymerase. There is a long untranslated region (UTR) at the 5' end that plays a vital role in translation, virulence and possibly encapsidation and a shorter 3' UTR necessary for the strand synthesis during the replication. The 5' UTR contains a 'clover leaf' secondary structure known as Internal Ribosome Entry Site (IRES) and the rest of the genome encodes a single polyprotein of around 2100 and 2400 Amino acid. The ends are modified by the attachment of VPg at 5' end and polyadenylation at 3' end as shown in figure. The structure (as in figure) which is icosahedral also reveals information about the assembly and function of mature capsid. Apart from VP1, VP2 and VP3, there is a small protein VP4 which is located predominantly on the inside of the capsid and not exposed on the surface. This VP4 is processed from polyprotein. These five VP4 monomers form a hydrophobic micelle, during the assembly of a pentameric subassembly. Also it is proved that these pentamers forms the vertices of the mature capsid (Cann, 2001).

In Picornavirus, penetration of the cytoplasm by the exit of virus from endosomes is linked to uncoating. The acid environment of endosomes changes the confirmation of the capsid which reveals the hydrophobic domains not present on the surface and the interaction of this domain with endosomes membranes helps the genome to pass into cytoplasm as shown in figure. The production of a polyprotein encompassing the whole of the virus genetic information is subsequently cleaved by proteases. These cleavages can be a subtle way of regulating the expression of genetic information. But in Picornavirus, the cleavages result in the production of various proteins with distinct properties from a single precursor (Cann, 2001). This is evident in figure.

replication of picornavirus rna

The purified genomic RNA is used to infect cells after the RNA is translated by the cellular ribosomes to produce viral proteins. This indicates that the structural protein present in the virion is not required to initiate the replication. The genome organisation of Picornavirus is explained in figure.

After the viral proteins have been made they proceed to replicate the viral RNA. It is also evident that P2 and P3 regions are involved in Picornavirus RNA replication. The replication starts with the synthesis of full length negative strand RNA with positive strand RNA as a template. These negative strands RNA is used as a template for the synthesis of addition positive strand RNA using multistranded replicative intermediates (figure). The number of positive strands synthesised in infected cells exceeds the negative strands in the ratio of 40:1. These viral RNA are formed in association with membrane vesicles that are required for virus infection and they require these vesicles as they serve as a nucleation site for the formation of replication complexes (Acheson, 2007). Once the viral RNA is synthesised the assembly of the Picornavirus virion involves cleavage of VP0 to VP2 and also VP4 (figure).

essential features of picornavirus ires elements

The secondary stem loop structure elements can be detected that indicate the presence of six stem loop structures (I to VI) in Enterovirus and Rhinovirus 5' noncoding regions. These structures are named type I and type II IRES. The 5' noncoding region of hepatitis A virus RNA share a similarity or homology with that of Picornavirus and this has been suggested to have a type III IRES elements. All Picornavirus IRES elements contain a pyrimidine rich strand 20-25 nucleotides from a conserved AUG codon. The distance between the pyrimidine rich track and conserved AUG is as important as their distance is involved in the initiation of translation. Additional conserved feature in Picornavirus IRES elements include a four ntGNRA sequence and rich sequences in specific loop regions. They play a role as they provide important information on RNA structure and 40S sequence required for binding of cellular proteins and 40S ribosomal subunit (Acheson, 2007).

Picornavirus Receptors:

The cellular receptors for several different groups of Picornavirus have been identified using a number of different techniques over the last few years (Rossmann, et al, 2002).:

Binding competition between different viruses

MAbs which block virus binding

Fluorescently labelled virus (Echovirus)

Virus:

# Serotypes:

Receptor:

Description:

Human Rhinovirus

91

ICAM-1 (Intracellular Adhesion Molecule 1, CD54)

Immunoglobulin-like molecule; 5 domains

Human Rhinovirus

10

LDLR (Low Density Lipoprotein Receptor)

Poliovirus

3

CD155

Immunoglobulin-like molecule; 3 domains

Coxsackie A

3

ICAM-1

Echo

2

VLA-2

Integrin-like molecule

Echo

6

DAF (Decay Accelerating Factor, CD55)

Also used by: CAV21, EV70

Regulation of complement activation

EMCV

1

VCAM-1 (Vascular Cell Adhesion Molecule, CD106)

Adhesion molecule

Table 1: Picornavirus and receptor interactions (Rossmann, et al, 2002).

Interaction of Picornavirus IRES elements with host cell proteins

It is predicted that the pre existing cellular proteins direct translation at IRES as there are no viral proteins other than VPg accompany Picornavirus RNA entry into a cell. The proteins that interact with IRES elements include La autoantigen, polypyrimidine tract binding proteins, poly (rC) binding protein 2 and the product of the unr gene. Although the precise roles of these proteins with IRES elements are known, the initiation factors are known for few of the cases. Also the cap independent translation utilises some eukaryotic factors that are involved in cap-dependent translation. The eukaryotic initiation factors that interact with IRES elements include eIF-2α and eIF-4B. The factors eIF-2, eIF-4E and eIF-4F help to stimulate cap-independent translation. The central portion of eIF-4G subunit replaces the function of eIF-4E and helps in the binding of eIF-4F. This recognises the cap and directs the complex to bon with the capped mRNAs (Acheson, 2007).

Picornaviruses make a variety of proteinases that cleave the polyprotein and some cellular proteins

Three distinct proteinases are encoded by Picornavirus, namely the L, the 2A and the 3C proteinases. Different genera have different cleavage pathways. The cattle disease, Foot and Mouth disease virus has as L proteinases, located at the very N terminus of the polyprotein. The L proteinase cleaves itself from VP4 protein and also cleaves cellular initiation factor eIF-4G, whereas other Picornavirus does not have L proteinases. The 2A proteinases of Enterovirus and Rhinovirus cleave at the P1/P2 junction and it is also responsible for cleavage of eIF-4G. But the 2A protein has no proteinases activity in other Picornavirus. One interesting fact is that all Picornavirus make the 3C proteinases. This cleaves at the specific dipeptides of the form glutamine-X, where X can be a glycine, serine or a number of other amino acids with hydrophobic side chains. These 3C proteinases exist in three different forms and each form has a different role in polypeptide cleavages. The initial cleavages of the nascent polyprotein are carried out by the cleaved 3C polypeptide in the intact polyprotein molecule. All these cleavages formed may be within the same polyprotein molecule or one 3C region can cleave within another polyprotein molecule. 3C cleaves at its own N-terminus within P3 to generate 3AB and 3CD. Further processing of the P1 region is carried out primarily by 3CD for Rhinoviruses and Enterovirus. A cellular protein Hsp 70, the molecular chaperone has been implicated in P1 processing. Also it is true that Hsp70 assists in the interaction between 3CD and P1. Alternatively, Hsp70 could help in the folding of P1 so that its cleavage sites are recognised by 3CD (Acheson, 2007).

Picornavirus proteins are made as a single precursor polyprotein that is automatically cleaved by viral proteinases

Many bacterial messenger RNAs contain several different protein coding regions and can INITIATE protein synthesis at various internal sites. Also in contrast, eukaryotic messenger RNAs code for single protein. This possess a problem for single stranded RNA viruses like Picornavirus to express a sing numerous proteins from a single strand RNA that serves both as a messenger RNA and genome. This is overcome by the linking of all of their protein coding regions into a single unit and synthesising a single, large polyprotein. This polyprotein in cleaved into different viral proteins by the act of proteinases. The initial cleavage that happens in the process is rapid. The polyprotein is first cleaved into three fragments called as P1, P2 and P3. P1 is cleaved into capsid proteins VP0, VP1 and VP3, whereas VP2 and VP4 are cleaved into a number of smaller proteins (Acheson, 2007).

Cell receptors for Picornavirus

It is found evident that 50% of all human common cold are caused by members of this Picornavirus. The cell surface receptors for these viruses are identified by screening the monoclonal antibodies. The amino acid sequence analysis of the protein obtained from the Rhinovirus of the Picornaviridae family identified it as an integral membrane intracellular adhesion molecule I (Icam-I). The natural ligand of Icam-I is another integral membrane protein known as Lymphocyte function associated antigen I (Lfa-I). The function of Icam-I is to bind to Lfa-I and to mediate a number of immunological and inflammatory responses. For other types of Picornavirus, one type of receptor is not enough so they include a cell receptor called as Decay-accelerating protein (CD55) (Flint, et al, 2009).

cleavage of VP0 to VP2 and VP4

Once the virus capsid proteins are synthesized and processed in the cells, the virion assembly involves the cleavage of VP0, VP1, VP2 and VP4. The process of assembling the virion starts after the sufficient amount of genomic RNA is synthesised. Once the protease P1 cleaved, the five protomers, each with a molecule of VP0,VP1 and VP3 assembles themselves to form a 14S pentamer structure (figure). Once the pentameric structure is formed, there are two ways of assembling the Virion. One is that, 12 of those pentamers coming together to form an 80S empty "Procapsid" and a single molecule of newly synthesized viral RNA threaded into the capsid to form a "Provirion". Then the final infectious virion is produced by the final proteolytic cleavage event that produces VP4 and VP2 from the VP0 precursor. Alternatively another mechanism is that the 14S pentamers associate themselves to the viral genome RNA and then combine with other 14S pentamers to form the Provirion directly. In either of the process only the positive strand viral RNAs with a VPg protein linked to the 5'end are packed. However the cleavage of VP0 carried out by the proteinases is not known. But it is evident that this cleavage is not carried out by the known viral proteinases. This is so, because the cleavage site is found inside the procapsids and provirions and would not be made accessible for the proteolytic enzymes to participate in the cleavage (Acheson, 2007).

Inhibition of host cell macromolecular functions

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. Protein 2A encodes for Enterovirus and Rhinoviruses and 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. Other Picornavirus inhibits cap dependent translation by binding and sequesteration of eIF-4E, the protein subunit of the eIF-4F complex that binds directly to the 5'cap structure of eukaryotic mRNAs. Also to make the point clear, mammalian cells were infected with Picornavirus that lead to the shutoff of host cell RNA synthesis. The foot and mouth disease in cattle, has a virus 3C proteinases which cleaves histone H3 which is a cellular protein associated with transcriptionally active chromatin in the cell nucleus. All these activities prove that the virus hinders the activity of the host cells and makes the host cell to devote all its metabolic activity to the process of replication and expression of Picornavirus in the host cells. Above all these issues, there is a notable virus, Hepatitis A virus which belongs to the family to Picornavirus that has a very slow replication rate, doesn't inhibit the host cell protein or RNA synthesis of the host cells. Also for the most part they do not produce observable cytopathic effects during the infection of cells in the culture (Acheson, 2007).

conclusion

It is all now evident that 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 drugs being designed now targets the proteinases that are being synthesized by the virus during the process of their replication. The drugs now act by hindering the growth of the Picornavirus by inhibiting the action of the proteinases of the virion.

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