Role Of Proteases In Picornavirus Replication Biology Essay

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The viral family Picornaviridae is made up of the genera Enterovirus Poliovirus and Coxsackievirus, Rhinovirus, Cardiovirus Theilers murine encephalomyelitis virus, Aphthovirus (Foot-and-mouth-disease virus), Erbovirus (Equine rhinitis B virus), Kobuvirus (Alchi virus), Teschovirus (Porcine teschovirus), Hepatovirus (Hepatitis A virus) and Parechovirus (Human parechovirus). This family of viruses represent very important human and animal diseases such as Polio myelitis, Hepatitis and Foot-and-mouth-diesease (Whitton et al, 2005). Infections from picornaviruses have very serious economic and health consequences to the world economy as a whole (Chase and Semier, 2012). Human rhinovirus cause the common cold which does not present with severe symptoms but costs billions of pounds in health care and missed worked annually (Cordingley et al, 1990; Jacobs et al, 2013). Foot and mouth disease caused by apthovirus which infects cattle, goat, sheep, pigs and other non-human animals, has led to the killing of over 2.5 million animals worldwide in outbreaks in China, Taiwan, Japan and the United Kingdom. Poliovirus causes poliomyelitis which affects the nervous system resulting in paralysis and muscle weakness and is suspected to have been around since ancient Egypt. Poliovirus vaccines were developed in the 1950s and have led to the near eradication of the disease (with occasionally cases being reported worldwide) (Nathanson and Martin, 1979; Dowdle and Birmingham, 1997). The importance of these viruses makes a greater knowledge of their architecture, infection and replication very important.

Just like every other virus family, the picornaviruses require the metabolic processes of the host cells they infect to replicate. In other to replicate, viruses must penetrate host cells and invade the intracellular environment of these cells. To achieve this, these viruses must interact with the intracellular cell environment and bypass their defences. (Agol and Gmyl, 2010). The picornaviruses have very effectively evolved to alter the cellular environment of the host cells to facilitate viral replication (Chase and Seimer, 2012). Due to the small size of the genomic RNA of these viruses and the limitation in the number of proteins produced, most of the proteins are multifunctional. If considered in this prospective, it is safe to think of the viral proteases as security proteins as proposed by Agol and Gmyl (2010) because they inhibit the host cell from synthesising new proteins while "hijacking" their replicative system to produce more viral particles.

A key feature of picornaviruses is a single stranded, positive sense RNA molecule of about 7500 nucleotides with a polyadenylated 3" end and a virus encoded protein, VPg protein at it 5" end (Stanway, 1990; Werner et al, 1986). The viral RNA has a very highly structured 5" noncoding region with stem loop structure (Chase and Semier, 2012). Picornaviruses are non-enveloped viruses with a capsid of about 25-35 mm. The capsid encloses a single-stranded RNA genome which is translated by a cap-independent mechanism after uncoating into a long polyprotein which is cleaved by the proteases L, 2A and 3C to produce structural and non-structural proteins (Stanway, 1990). After infecting a cell, the ribosomes of the host cell translate at a single site of the viral genome leading to the production of a large polyprotein with a molecular weight of about 250000 (Werner et al, 1986). An intriguing feature of the picornaviruses is their ability to replicate independent of the host cell transcription mechanism. This makes them a suitable model for studying the regulation of translation in eukaryotes and posttranslational modification (Strebel and Beck, 1986). Picornaviruses encode their RNA in a long open reading frame (ORF) flanked by long untranslated regions (UTRs) and the arrangement of the translated proteins is highly conserved through all the members of the picornavirus family (Whitton et al, 2005). In other to replicate, the viruses produce proteins that alter the machinery of the host cell such as protein localization, gene expression and membrane rearrangement (Lin et al, 2009).

The initial cleavage of the polyproteins produces the products L, P1, P2 and P3 (Strebel and Beck, 1986). The P1 region of the viral genome encodes the capsid particles which consist of 60 copies of the VP1-VP4 polypeptides coded for by the P1 region. VP1, VP2 and VP3 are located on the outer surface of the capsid while VP4 resides on the inner surface. These proteins are essential in host infection as they are responsible for receptor binding with the host cell membrane (Lin et al, 2009). The receptor molecules in picornaviruses are from the immunoglobulin superfamiy (IgSF) which have two-five amino-terminal immunoglobulin domains in their extracellular regions. The amino terminal domain in all these receptors, D1, binds with the conversed amino acids residues of the virus canyon which activates the uncoating of the virus. The rest of the genome encodes the non-structural replicative proteins of the picornaviruses.

Figure 1: Schematic of the enterovirus genome, the polyprotein products and their major functions. A diagrammatic representation of the enterovirus genome is shown. The 11 mature polypeptides are shown, together with the three main cleavage intermediates. The main biological functions are included for each polypeptide. UTR, untranslated region; IRES, internal ribosome entry site; VPg, viral protein genome-linked (Lin et al, 2009).

Alternations in host cell membrane permeability occur during viral infections at defined times; when the viral particles penetrate the cells and when new viral particles are being synthesised. This permeability change is important to allow "injection" of the viral RNA and the release of newly formed particles. The pathways involved in this process poorly understood (Aldabe et al, 1996). These alterations can be seen by an increase in cytosolic free calcium concentrations in infected cells. Aldabe et al (1996) suggest that the concentration of stored and free calcium increases during a viral life cycle indicating that calcium is needed for the production of viral particles.

Picornaviruses encode their proteins in a single open reading frame and contain three types of proteinase-L, 2A and 3C (Ryan and Flint, 1997). The viral RNA is expressed by direct translation into a large polyprotein precursor (Allaire et al, 1994). . The 2A and 3C proteases are virus encoded proteins which play an important role in polyprotein processing.

The development of autoproteolysis by viruses has enabled then to regulate protein production by encoding their own proteases instead of relying on the host cells for proteolytic processing (Ryan and Flint, 1997). Picornavirus proteinases are essential in the cleavage of the polyprotein into its functional components (Blom et al, 1996). The different identified picornaviruses display variations in the complex cleavage pathways of their proteins (Rueckert and Wimmer, 1984). After RNA translation, a polyprotein is produced which is cleaved in three (in the case of poliovirus) or four (in the case of foot and mouth disease virus) primary proteins. Picornavirus polyproteins contains the L, 2A and 3C proteinases (Ryan and Flint). The primary proteins are then cleaved by the viral proteases into functional proteins (Burroughs et al, 1984). The polyprotein is rapidly and highly modified after translation by these proteases to produce the replicative proteins. These proteins are responsible for the posttranslational cleavage of the polyprotein into mature proteins which are packaged and released to infect other cells.

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Figure 2: Schematic representation of primary proteolytic processing in six picornavirus genera (Seipelt, 1999). The L protein of cardiovirus is not a proteinase unlike in the aphthovirus (Ryan and Flint, 1997).

L: The L and 2A proteinases vary through the picornavirus family. L varies from a length of about 70 amino acid residues in cardiovariuses to about 450 amino acid residues in some sapeloviruses. (Agol and Gmyl, 2010). The leader proteinase, L(pro) protein is unique to the aphthoviruses and the equine rhinoviruses and is located at the N terminus of translated polyprotein (Ryan and Flint). The L(pro) of the aphthoviruses is a paparin-like cysteine proteinase (Foeger et al, 2002). Cells infected with The foot and mouth disease virus experience an inhibition of protein synthesis as a result of cleavage of the translation initiation factor Eif4a by L(pro) (Belsham et al, 1999). The L protein releases itself from the polyprotein although in cardioviruses and kobuviruses, the L protein is non proteolytic and is released from the polyprotein by 3C (Whitton et al, 2005). The L proteins cleaves at its own C terminus and has been suggested to exist in at least two forms; Lab(pro) and Lb(pro) where the Lb(pro) undergoes posttranslational modification producing Lb'. Both forms however are capable of cleaving the L/P1 junction (Ryan and Flint, 1997). Aside from self-cleavage from the nascent polyprotein, L protein also cleaves the host cell protein Eif4G (just like 2A) by acting between Gly and Arg residues of the protein. L(pro) is the first protein synthesised in the polyprotein and does not directly play a role in viral replication. This protease also cleaves Eif4g (just like 2A and 3C) (Castello, 2011). Because this protein is not produced in all picornaviruses, it is of particular interest as its full potential and activity is not fully understood.

2A: The 2A protease is about 17 kDa in size and cleaves the polyprotein at its own N terminus. Primary cleavage in enterovirus and rhinovirus occurs between the P1 protein precursor and the replicative domains of the polyprotein (P2 and P3). In poliovirus 2A cleaves at the P1-P2 site of a segment of the polyprotein (Krausslich et al, 1987) at a tyrosine-glycine scissile pair (Ryan and Flint, 1997). The 2A proteinase cleaves at the site between the capsid precursor protein P1 and the non-structural P2-P3 region at its own N-terminus (Blom et al, 1996) and this represents the initial cleavage of the polyprotein (Stanway, 1990). A host cell infected with the human rhinovirus or enterovirus has a compromised ability to translate capped mRNA while allowing the production of viral RNA. This phenomenon is a form of host modification called host cell shutoff (Liebig et al, 1993). It is suspected that the 2A protein in apthoviruses and cardioviruses might not be proteolytic and show a conserved Asn-Pro-Gly-Pro motif at the 2A-2B junctions which might be cleaved by ribosomal skipping and not 2A (Whitton et al, 2005). Another function of 2A(pro) is the ability of inhibiting protein synthesis by the host cell by cleaving Eif4g which is associated with cap binding protein complex resulting in host shut off. However, in aphthoviruses and cardioviruses, the primary cleavage occurs not at the N terminus, but at the C terminus of the 2A region between the capsid protein P1-2A and 2B. In hepatoviruses, this primary cleavage separating the capsid proteins from the replicative proteins is not carried out by 2A but rather by 3C.

3C: The picornavirus proteinase 3C(pro) is a cysteine proteinase with a trypsin-like polypeptide fold (Allaire et al, 1994; Hammerle et al, 1991; Mattews et al, 1999) which is found in all picornaviruses at a very high degree of similarity. This proteinase has a molecular weight of about 20 kDa which folds into two equivalent six stranded B-barrels with a groove for substrate binding (Matthews et al, 1999). 3C mediates primary cleavage between 2C and 3A and is responsible for a number of secondary cleavages leading to the production of replicative precursors. It accounts for most of the cleavage of the polyprotein into mature proteins and occurs between Gln-Gly pairs. It has been reported that 3C can translocate into the nucleus through its precursor 3CD where it cleavages regulators involved in DNA dependant RNA polymerase I, II and III such as TATA-box binding protein, octamer binding protein, cyclic AMP responsive element binding protein and DNA polymerase III (Amineva et al, 2004; Clark et al, 1991; Clark et al 1995; Lin et al, 2009; Yalamanchili et al, 1997). The 3C precursor protein 3CD in poliovirus circulates the viral genome by interacting with the 3" and 5" ends of the viral RNA. An added activity of 3C is the cleavage of host cell proteins such as histon H3 and microtubule associated protein 4 (Ryan and Flint, 1997). 3C also cleaves the poly(A) binding protein (PABR) at three sites; the linker region and two regions between the RRM region. The PABR which is normally involved in Mrna circularization for the initiation of translation is cleaved by 3C in infected cells, leading to interference in the cap dependent translation in host cell proteins (Chase and Seimer, 2012).

It has also been demonstrated that the foot and mouth disease virus is able to counteract the innate immune system of the infected animal. As described by Wang et al (2012), the innate immune response is activated by host recognition receptors recognising pathogen associated molecular patterns (PAMPs) afterwhich the recognition receptors trigger the production of type 1 interferons and inflammatory cytokines. By a pathway which involves the activation of various cytokines and factors including MDA5, RIG-1 and NF-KB, the immune system is able to recognise and eliminate infected cells. Their research suggests that 3C impairs signalling by RIG-1/MDA5 by cleaving the downstream adaptor protein, NEMO. This leads to greater survival of the viral infected cells.

Strebel and Beck (1986) suggested that the principle used by these viruses to produce functional genes is the regulation of gene activation by proteolytic cleavage of the translated polyprotein. The activities of these proteases are essential in the successful infection and replication of the picornaviruses. The production of the mature proteins from the posttranslational modifications leads to the release of newly formed viral particles which go on to infect other cells. Due to the s limitation in the proteins produced, it is important that these proteases are conserved in the genome of the viruses. The added property of these proteases in mediating host shut off provides an added benefit to the virus as the host machinery is manipulated to facilitate viral replication. Without these proteases, the viruses would be unable to replicate and are a target for drug development. The proteolytic activities of these viruses could also provide insight into the molecular level machinery driving viral replication and other eukaryotic systems as a model for other organisms. Ryan and Flint (1997) stated that the ability of viruses to encode their proteases within the polyprotein eliminates the potential for temporal control of the protein expression by transcription. This method conserves the production of these necessary proteases. According to Chase and Semier (2012), the ability of picornaviral proteases to not only cleave the translated polyprotein but also disrupt host cell transcription is advantages to viral replication as these viruses are strictly cytoplasmic. This means that disrupting the nucleocytoplasmic "shuttling" of the host cells ensures the efficient production of viral proteins.