The Role of Proteolytic Activities in Picornavirus Replication

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There are several viruses with their history dating back to the ancient times. Of such known ancient viruses, the Picorna virus is one. An evidence of its origin to old times is the discovery of a few Egyptian carvings known to date back to 1400 B.C. illustrating symbols of polio in man. Regarding the discovery of this virus, it is said that Loeffler and Frosch discovered FMD virus in 1898 while Landsteiner and Popper found Poliomyelitis as a viral in 1909. The name of Picornaviruses is Latin where 'pico' refers to small and thereby these viruses are small with a positive sense RNA genome, single stranded with no envelope and having an icosahedral symmetry. Picronavirus has a diverse variety of hosts and infections but there is no clear evidence about their pathophysiology and gene expression system. In humans as well as animals, this virus causes various diseases from the severity of infections ranging from sub-clinical mild ones to chronic liver and cardiac ones. The virus belongs to the family picornaviridae which is a large viral family with 8 genera and 3 proposed genera: enterovirus, cardiovirus, aphthovirus, hepatovirus, parechovirus, erbovirus, kobuvirus and teschovirus; sapelovirus, senecavirus and tremovirus (proposed). This virus has over 200 serotypes and every genera has various viruses that result in different infections. (1)(2)

It is only be means of pathegenic studies that scientists are able to differentiate genera that are identically structured. In majority of the types, genome structure and arrangement is identical but there is presence of some variances that will be later explained comprehensively in one of the following sections. In the research about viruses, virus genetic system is an essential element and therefore in viral pathegencity and clinical epidemiology, genome arrangement and replication are considered to be primary features. According to classification scheme of Baltimore, Picornavirusues fall under group IV viruses and they exist in varying sizes of 1-30nm. They have no lipid envelop and show properties of resistance towards ether, chloroform and alcohol. Radiation, formaldehydes and phenol groups are a few of the familiar means by which they can be killed. It is said that genome organization and its replication are crucial elements in viral pathegencity and clinical epidemiology and thus in the virus studies, their genetic mechanism are important. Pathegenic studies are used for differentiating identically structured genera. In this virus, majority of the types have similar genome structure and organization with some variations that are later explained comprehensively. (3)

GENOME ORGANIZATION AND FEATURES

Genome is enclosed in icosahdral capsid comprising of 60 copies of VP1, VP2, VP3, VP4 proteins derived from the protomer VP0. The capsid performs various important tasks such as RNA protection, 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 phosphates of nucleic acid. The genome is around 2500nm length, with a view from 5' end to 3' end, similar to eukaryotic mRNA, has positive sense RNA molecule which is 7.2kb to 8.5kb and comprises single strands. As the RNA is positive sense, it can also perform as mRNA where the exposed genome has the ability of infecting the cells with lesser virulence than particle of the virus. It is possible to raise the infectivity by transfecting the RNA into host cells. At both ends, it has complex structured untranslated regions (UTR). The UTR at the longer end is approximately 600-1200 bp and performs a key part in viral translation, virulency and encapsidation while that at the shorter 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 between 2100-2400 AA sizes. At the 5' end, there is a basic VPg protein and an 'internal ribosome entry sites' (IRES) which is a secondary structure similar to 'clover leaf'. IRES is a unique characteristic of picornavirus mRNA and helps to manufacture protein in infected cells irrespective of grave alterations in translation initiation elements stimulated by viral proteases and leading to hindrance in manufacture of cellular protein. The 3' end is also polyadenylated. Thus both ends have auxiliary modification. (4)(5)(6)

THE POLYPROTEIN AND INDIVIDUAL PROTEINS

Another aspect worth including in this discussing is the aspect of polyprotein. 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 particular places where the proteins are present on the polyprotein. The only proteins that are involved in replication are VP1-4 which is a structural protein with a place in capsid structure but the rest are functional protein and accompanied by their precursors have particular roles and are crucial for the replication inclusive of nucleic acid replication, 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. There is vagueness when it comes to the precise features and system of the VP0 cleavage with VP4 and VP2, also known as the maturation cleavage but the particle assembly and stability is not possible without it. A discussion on their individual roles is vital in this essay's framework, particularly related to replication. 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 of host cell. 2A has a different structure but is otherwise identical with varying sizes and functions in various viruses. In serves as protease similar to trypsin in enterovirus while it serves as a processor in aphthovularirus and cardiovirus. It helps in replication of cell and proliferation by means of interference in cell cycle in case of parechovirus, kobuvirus and tremovirus. It is not possible to initiate polyprotein processing and genome replication without it. Modification on 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 3' NCR of negative sense RNA. 3A also participates in genome replication as it relates to 5' and 3' both UTRs. It is also suggested that 3A inhibits endoplasmic reticulum to Golgi complex transport in the cell and by membrane association; 3AB plays a part in RNA replication. At the 5' end, there is 3B or VPg which is said to be replication's protein primer. The primary proteases are 3C and 3CD in the entire arrangement and cause a large number of processing events even though they are multi-functional and attached to the nucleic acid. To replicate and elongate RNA, 3D is the viral RNA-dependent RNA polymerase which is required. Protein L thus has varying functions in various species. The following section will further discuss how these proteins take part in replication. (6)(7)(8)(9)

Fig 1) This figure illustrates the process of how the genome performs encoding on a single polyprotein. This polyprotein is then processed for creating mature individual proteins.

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

REPLICATION

Picornavirus replication takes place entirely in the cytoplasm of 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

When it comes to binding of the receptor in picornavirus, an important feature is that every picornavirus has a distinct receptor. Though it is said that majority of these viruses utilize surface molecules falling under the umbrella of immunoglobulin superfamily (IgSF) and 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. Thus attaching itself to a certain receptor is the first step in the process. (10)

Un-coating

VP4 release is a part of uncoating stage. De-stabilization of viral capsid occurs post to the binding, this then results in release of VP4, allowing the viral RNA to penetrate the host cell where uncoating of the RNA takes place and its replication is done by means of double stranded RNA intermediate. Protein manufacture and viral nucleic acid replication both takes place from this viral genome. The double strand results from the use of 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.

IRES located at the 5' end of the genome, also called landing pad, starts translation. By means of cap-independent mode, IRES attaches to the ribosome precisely without the need to scan and transfers it to the polyprotein initiation condon. Majority of the time, translation is started by the attachment of ribosome to the 5' methylated cap and a subsequent scan of mRNA to search for the first initiation condon. Cleavage of eIF-4G protein does not permits normal mode of translation of cellular genes after it has been infected with picornavirus but it is immune to IRES driven translation. This is an effective manner for the virus to keep the host cell from protein manufacturing to varying extent and keep on with its procedure. This is possible as a result of cellular protein eIF-4G, a part of 220KD 'cap binding complex' cleavage, facilitated by 2A proteinase in enteroviruses and rhinoviruses by cleaving 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 resulting in cleavage of eIF-4G part of the eIF-4F eukaryotic translation initiation complex vital for cap-dependent translation. Almost every host gene is blocked by polioviruses while merely half of it is blocked by rhiniviruses. (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' end non coding region along with various 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. These are some causes for the picornaviruses to utilize the non-conventional cap-independent process. There is also transfer of cell machinery of the host cell to viral proteins machinery during polivirus infections as human rhinovirus or coxsackevirus, the viral proteinases including 3C and 2A result in various proteins binding in the cell inclusive of initiation factor eIF4G. This consequently leads to shutting off of cap-dependent translation. (14)

The coding of viral genome takes place only in certain regions. P1, P2 and P3 are the regions of viral genome's coding. The structural and functional proteins come from these regions 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 replication of itself come from P2 region while P3 region provides those required for picornavirus replication such as 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 binded by 3Cpro in cardiovirus and aphthovirus infected cells where the 2A proteins of such virus merely perform catalysis of their own discharge 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 andparechovirus but VP4 is the discharge catalyst for FMD virus. (11)(15)

There is co-transition in the initial events and it commences when translating ribosome still has virus RNA and polyprotein attached to it. This also presents an explanation regarding why the infected host cells do not have the complete length polyprotein. Structural as well as non-structural viral proteins are generated from the proteolytic processing of the viral polyprotein by proteinases like L, 2A, 3C and 3CD. Aphthovirus encodes Proteinase L, entero-and rhinovirus encodes 2A while almost all picornaviruses encode 3C and 3CD. 2A proteinase has a cysteine nucleophile with an identical structure to chromotrypsin-like proteinase B and mediates primary cleavage events at the P1 and P2 junction. The main site for cleavage events is N-terminus of 2A proteinase and this leads to P2-P3 precursors and polyprotein P1 region. Cleavage of viral protein results from the folding of 2A into an active conformation in the course of translation. L-P1 junction is the primary site in aphthovirus leading to L protein discharge from the viral protein's N-terminus. L proteinase is responsible for primary cleavage in aphthovirus. In cardiovirus, L protein is distinct from L protein of earlier mentioned groups and performs no proteinase activity. 3C proteinase is important for P1 precursor to result in L protein cleavage in cardiovirus with NPGP peptide mediating it from 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 hold the prime responsibility of execution and this also result in crucial elements to process protein and in genome replication. 3C 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 cysteine in 3C proteinase with an identical structure as protease chymotrypsin. It plays an important part in processing events after it has undergone self cleavage from P3 precursor protein leading to P2 and P3 precursor generation of replication proteins. It is said that in a few poliovirus infection, C and 3CD cleave the poly (A) binding protein which causes the translation in process to inhibit. It is also assumed that it will result in various host cell proteins cleavage that are linked with mamalian cell pol 1, pol II and pol III transcription. (14)(18)

GENOME REPLICATION

Following section discusses the various protein activities involved in genome replication as in translation:

Proteins are crucial in the replication process of the picornavirus. Picornavirus has a distinct genome replication system comprising of various activities by each protein. At the 5' end of the genome, VPg peptide is covalently linked with picornavirus using a process based on multiple steps. Viral precursor protein 3CD or its processed derivative 3C greatly influences the following reaction and complete genomic RNA results from using VPg-pU-pU as a primer. Uridylylation process yields VPg-pU-pU, a uridylylated VPg coming into existence from Tyr-3 of VPg which is acting here as a primerby the viral RNA depemdent RNA polymerase (3Dpol). Interaction with 3C as well as 3CD may be required and necessary when the concern is 3Dpol and its stability. It is true that picornavirus genome replication cannot take place without VPg-pU-pU creation but there is no consensus about a template for VPg uridylylation. Besides, in VPg uridylylation, there is confusion between oril or poly(rA) 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. 3C protease and 3D polymerase domain combine to form protein 3CD which thus shows evident signs of specific protease activity, which is a catalytic activity dissimilar to 3C. 3CD has no signs of 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 3C domain without any part played by 3CD. The initiation site for manufacturing of negative strand RNA is the viral 3' poly (A) tract post to the creation of VPG-pU-pU. Positive strand RNA genomes are produced with the help of negative strand template which are later converted into virions or at times even act as template for manufacturing of viral proteins. (19)(20)

ASSEMBLY AND MATURATION

Enclosing the viral genome in capsid is the final step in the process that has been under discussion. 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. Majority of the time cell lysis discharges virus from host cell except in the case of only a few viruses like that of Hepatitis A which is assumed to belong to less lytic variety. Despite the studies that have been undertaken, there is ambiguity regarding the precise packing and genome interaction with the capsid.

Fig 2) This figure shows the entire cycle of replication in Poliovirus. This figure also presents the proteins' 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, we have reviewed various characteristic 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 shows how essential proteolytic activities are in picornavirus replication and the replication will not be possible without proteolysis of the viral polyprotein. Another important thing to be observed is that 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 too. 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 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 it can be controlled from replicating in various kinds of hosts is much deeper and wider than it was earlier considered to be. In order to control or stop the virus from harming the hosts, it is important to understand how to restrict its replication. Therefore, scientists ponder upon the point where replication can be halted in a host cell and what are those proteins whose blocking can achieve this objective. 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.

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