Role Of Proteolytic Activities In Picornavirus Replication Biology Essay

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Egyptian carvings from 1400 B.C. illustrate the symptoms of polio suggesting that picornaviruses have been infecting humans throughout recorded history. Loeffler and Frosch discovered the FMD virus in 1898, while Poliomyelitis was isolated by Landsteiner and Popper in 1909. The name picornavirus is derived from 'pico' meaning small and the naked single stranded, icosahedral RNA, which constitutes their genome. Picronaviruses have a diverse variety of hosts and infections but there is no clear understanding of their pathophysiology and gene expression system. In humans as well as animals, this virus causes diseases ranging from the sub-clinically mild to chronic liver and cardiac conditions. The picornaviridae is a large viral family with eight established genera and three proposed genera including enterovirus, cardiovirus, aphthovirus, hepatovirus, parechovirus, erbovirus, kobuvirus and teschovirus; sapelovirus, senecavirus and tremovirus (proposed). The family has over 200 serotypes and a wide variety of disease causing members. (1)(2)

Pathogenic studies allow scientists to differentiate genera that are identically structured. In the majority of the picornaviridae, genomic structure and arrangement is identical with some genetic variance as explained below. According to the classification scheme of Baltimore, picornaviruses are group IV viruses around 1-30nm in size. They have no lipid envelop and show resistance to ether, chloroform and alcohol. They are sensitive to radiation, formaldehydes and phenol groups. (3)

GENOME ORGANIZATION AND FEATURES

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The genome is enclosed in an icosahedral capsid comprising 60 copies of the VP1, VP2, VP3 and VP4 proteins derived from the protomer VP0. The capsid protects the RNA, and has roles in determination of host and tissue tropism, target cell penetration, RNA transfer and viral RNA packaging. Sodium and potassium ions are also present in the capsid for neutralizing the negative charge present on the phosphates of nucleic acids. The genome is around 2500nm in length, and is a single-stranded, positive sense RNA molecule (coding 5' end to 3' end, similar to eukaryots) of 7.2kb to 8.5kb. As the RNA is positive sense, it can also act as mRNA and giving secondary virulence in host cells. It is possible to raise the infectivity by transfecting the RNA into host cells. At each end it has complex untranslated regions (UTR). The UTR at the longer end is approximately 600-1200 bp and performs a key part in viral translation, virulence and encapsidation while the other end is around 50-100 bp with a part to play in negative strand synthesis during replication. The entire genome encodes for a single polyprotein of about 2100-2400 amino acids. At the 5' end, there is a basic VPg protein and 'internal ribosome entry sites' (IRES), which form a secondary structure similar to a clover leaf. The IRES is a unique characteristic of picornavirus mRNA that aids in protection from viral proteases and leads to hindrance in manufacture of cellular proteins. The 3' end is polyadenylated, thus both ends have auxiliary modifications. (4)(5)(6)

THE POLYPROTEIN AND INDIVIDUAL PROTEINS

The polyprotein is broken up by a proteolytic series of events, mediated by virally encoded enzymes, into precursors and then discrete proteins. It is important to discuss genome coding of single protein in the context of viral replication. A proteolytic series of events related to viral encoded enzymes processes and breaks down the polyproteins into individual proteins in the order of breaking down first of the precursors and then breaking down of the discrete proteins. Equal importance will be given to detailed discussion about the polyprotein organization and the tasks which are performed by individual proteins as each of the protein included in polyprotein has its own characteristics.

L, 1A (VP4), 1B (VP2), 1C (VP3), 1D (VP1), 2A, 2B, 2C, 3A, 3B (VPg), 3C, 3D are some of the proteins present on the polyprotein (Fig. 1). The only protein involved in replication is VP1-4, which is a structural protein with a place in the capsid structure. The rest, with their precursors, are crucial for the replication of nucleic acids, polyprotein processing and other purposes. VP1-3 is identical in size and basic structure to VP1-4 but much larger and present outside the capsid. VP1-3 needs VP0, the precursor, for assembly of the particle. The precise features and system of the VP0 cleavage with VP4 and VP2, also known as the maturation cleavage, are unclear, but particle assembly and its stability are not possible without it. Protein L serves as a protease in aphthovirus and erbovirus while it helps to move proteins and cytoplasm in cardiovirus, kobuvirus and teschovirus. It also terminates translation processes of the host cell. 2A serves as a protease similar to trypsin in enterovirus and as a processor in aphthovirus and cardiovirus. It assists replication of the cell and proliferation by means of interference in the cell cycle in the case of parechovirus, kobuvirus and tremovirus. It is not possible to initiate polyprotein processing and genome replication without it. Modifications of the host cell membrane and release of virions by host cell lysis is facilitated by 2B. 2C and its precursor play a part in RNA replication by binding the precursor to the 3' non-coding region of negative sense RNA. 3A also participates in genome replication as it binds to both 5' and 3' UTRs. 3A may also inhibit endoplasmic reticulum to Golgi complex transport in the cell by membrane association. 3AB plays a part in RNA replication. At the 5' end, 3B, or VPg, may be the replication protein primer. The primary proteases are 3C and 3CD and are involved in a large number of processing events even though they are multi-functional and attached to the nucleic acid. 3D is the viral RNA-dependent RNA polymerase required to replicate and elongate RNA. Protein L has varying functions in various species. The following section will further discuss how these proteins take part in replication. (6)(7)(8)(9)

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Fig 1) The picornavirus polyprotein is processed into individual proteins.

Source : www.expasy.ch/viralzone/all_by_protein/33.html

REPLICATION

Picornavirus replication takes place entirely in the cytoplasm of the host cell. Various in depth system of replication are available in documentations, and these documentations have come into existence as a consequence of the hard work of scientists that they placed into the study of picornavirus replication. The process is dependant on the viral precursor proteins' proteolysis and can even take place in cells with no nucleus. The following steps define the process:

Receptor Binding

Every picornavirus targets and binds to surface molecules of the immunoglobulin superfamily (IgSF). Most of the rhino- and enteroviruses employ a feature similar to canyon for binding the receptor and this eventually facilitates un-coating at an early point in infection. (10)

Un-coating

De-stabilization of the viral capsid occurs after binding, which results in release of VP4, allowing the viral RNA to penetrate the host cell where uncoating of the RNA takes place and replication occurs by means of a double stranded RNA intermediate. Protein manufacture and viral nucleic acid replication take place during this double strand phase mediated by viral RNA dependant RNA polymerase. (11)

Translation

Various crucial proteolytic steps comprise viral translation and in this section discussion will be done on every one of it as it occurs in the entire cycle of replication..

The IRES located at the 5' end of the genome, also called the landing pad, starts translation. In a cap-independent manner, IRES attaches to the ribosome precisely without the need to scan, and transfers it to the polyprotein initiation codon. This is in contrast to normal translation, which occurs by attachment of the ribosome to the 5' methylated cap and a subsequent scan of mRNA for the initiation codon. Cleavage of eIF-4G protein blocks normal translation of cellular genes after infection by picornavirus, which effectively disables the host cell from manufacturing its own protein, but it allows IRES-driven translation. There is no 7-methylated G cap structure in the genome of picornavirus though it is usually present at the 5' end of cellular mRNAs, and it is needed for cap-binding complex recruitment. 2A proteinase, in enteroviruses and rhinoviruses, cleaves off the eIF-4G part of the cap-binding complex during infection. In aphthoviruses, the barrier is a consequence of L proteinase, which stimulates the same host cell barrier by cleaving the eIF-4G part of the eIF-4F eukaryotic translation initiation complex that is vital for cap-dependent translation. Almost every host gene is blocked by polioviruses, while around half are blocked by rhinoviruses. (12)(13)

There is no 7-methylated G cap structure in the genome in picornavirus though it is usually present at the 5' end of cellular mRNAs which are needed for cap binding complex recruitment. There is also the presence of 5' end5' non-coding region along with various has several upstream non-authentic start codons. Thus, this process is only used where there is recognition of an appropriate sequence, RNA structure or ribonucleoprotein complex by the 40S ribosomal subunit. There is also transfer of cell machinery of the host cell to viral protein manufacture during poliovirus infections, human rhinovirus or coxsackievirus. The viral proteinases, including 3C and 2A, result in additional proteins binding to factor eIF4G, leading to inhibition of cap-dependent translation. (14)

The coding of viral genome takes place at P1, P2 and P3, where the structural and functional proteins are present in varying percentages. P1 gives 75% of the structural proteins while P2 and P3 provide functional proteins. Proteins vital for membrane reorganization of infected cells and virus self-replication come from the P2 region while the P3 region provides viral proteinase 3C and RNA dependent RNA polymerase 3D. The viral proteinases 2A and 3C (including 3Cpro and 3cdpro) are responsible for the primary cleavage activity. The binding of P1 and P2 is different in various infected cells. P1 binds to P2 by 2Apro in cardiovirus and aphthovirus infected cells while P1 and P2 are bound by 3Cpro in cardiovirus and aphthovirus infected cells where the 2A proteins catalyse their own product at the junction with 2B instead of acting as proteinases. 3Cpro also performs binding of 2A and 2B in certain types, such as hepatovirus and parechovirus, but VP4 is the discharge catalyst for FMD virus. (11)(15)

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There is co-transition in the initial events and it commences when the translating ribosome still has viral RNA and polyprotein attached to it. This also explains why infected host cells do not contain the full length polyprotein. Structural and non-structural viral proteins are generated from the proteolytic processing of the viral polyprotein by the proteinases L, 2A, 3C and 3CD. Aphthovirus encodes Proteinase L, entero- and rhinovirus encode 2A while almost all picornaviruses encode 3C and 3CD. 2A proteinase has a cysteine nucleophile with an identical structure to chymotrypsin-like proteinase B and mediates primary cleavage events at the P1 and P2 junction. The main site for cleavage events is the N-terminus of 2A proteinase and this leads to P2-P3 precursors and the polyprotein P1 region. Cleavage of viral protein results from the folding of 2A into an active conformation during translation. The L-P1 junction is the primary site in aphthovirus that leads to L protein production from the viral protein's N-terminus. L proteinase is responsible for primary cleavage in aphthovirus. In cardiovirus, L protein, uniquely, performs no proteinase activity. 3C proteinase is important for P1 precursor to result in L protein cleavage in cardiovirus with mediation by the NPGP peptide of the 2A coding region. (14)(16)(17)

With respect to the secondary cleavage events, each protein has its own role to play in the process but 3C proteinase and its precursor 3CD are primarily responsible. 3C also performs a few vital key processing events in viral protein processing while its precursor 3CD performs more proteinase activity than mature 3C. The active site nucleophile is a cysteine in 3C proteinase with an identical structure to protease chymotrypsin. It plays an important part in processing events after it has undergone self cleavage from P3 precursor protein to the P2 and P3 replication proteins. In some poliovirus infections, C and 3CD cleave the poly (A) binding protein which inhibits translation; it also affects mammalian pol 1, pol II and pol III transcription. (14)(18)

GENOME REPLICATION

Picornavirus has a distinct genome replication system comprising distinct proteins. At the 5' end of the genome, VPg peptide is covalently linked with picornavirus using a multi-step process. Viral precursor protein 3CD, or its processed derivative 3C, greatly influences the subsequent reaction and complete genomic RNA results from the VPg-pU-pU primer. Uridylation yields VPg-pU-pU, a uridylated VPg derived from Tyr-3 of VPg which is acting here as a primer for the viral RNA dependent RNA polymerase (3Dpol). Interaction with 3C and 3CD may be required and necessary for 3Dpol and its stability. Picornavirus genome replication cannot take place without VPg-pU-pU but there is no consensus about a template for VPg uridylation. In VPg uridylation, there is confusion between the poly(A) tail and creation of genomic or antigenomic RNA. A 'slide back' system is linked with VRg-pU-pU whereby oril, which is a single adenylated residue in an RNA stem-loop structure, acts as template. The 3C protease and 3D polymerase domain combine to form protein 3CD which has specific protease activity, and is a catalytic activity dissimilar to 3C. 3CD has no polymerase activity despite the fact that the general fold of the 3D domain of 3CD is quite similar to 3Dpol. RNA-binding and protease activities are performed by 3C and 3CD both; oril is encoded by the former while its affinity is improved by 3D. Specific and non-specific binding activity is shown solely by the 3C domain without any part played by 3CD. The initiation site for negative strand RNA is the viral 3' poly (A) tract after manufacture of VPG-pU-pU. Positive strand RNA genomes are produced with the help of negative strand template. These are later converted into virions or at times even act as templates for manufacture of viral proteins. (19)(20)

ASSEMBLY AND MATURATION

Enclosing the viral genome in its capsid is the final step in the process. Cleavage of P1 polyprotein precursor and VP0, 1 and 3 assemble the capsid and the subsequent enclosure of the genome within the capsid then takes place. Cell lysis is the usual method of viral discharge from the host cell except in the case of a few viruses like Hepatitis A, which is assumed to belong to less lytic variety. Despite much research, there is no consensus regarding precise packing and genomic interaction with the capsid. Figure 2 summarises the replication of poliovirus.

Fig 2) Replication in Poliovirus. This figure also presents the protein arrangement on the genome and how proteolysis takes place in the host cell cytoplasm.

Source: www.nature.com/.../n6/images/nrmicro906-i2.gif

Conclusion

In this paper, I have reviewed the characteristics of the picornaviruses, the mechanism by which they replicate and the importance of various types of proteins in the various stages of replication. The above discussion highlights the importance of proteolytic activities in picornavirus replication as replication is not possible without proteolysis of the viral polyprotein. Importantly, in the viral replication cycle individual protein generation by means of polyprotein processing is crucial and produces not only structural but also some functional proteins. The primary process in the cycle is genome replication and each protein has a vital part to play in this process. The primary implication and the purpose of replication is thus the production of more particles of the virus, and this very purpose will be void when the process will stop when no structural proteins are created and there is no transcribing and translation in the virus genome. The viral process halts in case that the genome replication takes place in an inappropriate manner. Minor variations in the proteolytic process takes place among various viruses during the process but the essence of the system remains important. It will be impossible for the virus to conclude its replication if there is no protolysis of the precursor polyprotein. It is equally important to remember that discharge of structural as well as functional protein will not occur if the viral polyprotein is remain intact and is not proteolysed.

Picorviruses is not limited in its effects and thereby it causes a large number of diseases of various levels of severity and affecting a number of organs and functions in various kinds of hosts. In the days long gone, the depth and influences of this virus was only limited and so was its understanding. With the recent studies, it has come to surface that the practical aspects of the comprehension of virus and the means by which picornaviruses can be controlled appears centred on its replication. Therefore, anti-viral drug designers will target points where replication can be halted in a host cell by what blocking the activity of crucial proteins.For exploring such dimension, a new field of medicine known as anti-viral drugs has been discovered by scientists with the aid of comprehension attained in the domain of what significance proteolytic activities and individual proteins have in the cycle of picornavirus.