Proteolytic Activities Involved In The Picornavirus Replication Biology Essay
The picornavirdae household ( picorna virus ) is known to do a wider scope of well surveies diseases, such as hepatitis, infantile paralysis and mycocarditis etc. as compared to other virus households. The unwellness caused by the viruses may be symptomless or do clinical syndromes. Foot and mouth disease is caused by picornavirus which was the first carnal virus to be discovered in 1898 ( Acheson, 2007 ) Poliovirus is the most extensively studied picornavirus for vaccinum development. These viruses are 7500nt in length and consist of a 3 ‘ poly ( A ) tail with variable length changing from 65-100nt. This virion RNA has a virus encoded peptide Vpg attached at its 5 ‘ end point. ( Racaniello et al. , 2006 ) . The term picorna can be split into pico intending really little ( 18-30nm ) and the RNA mentioning to the individual stranded positive sense RNA that the virus household possesses.
GENOME OF PICORNAVIRUS
The genome of picornavirus contains an ssRNA ( + ) . The nucleic acid RNA ( + ) nowadays in picornavirus in considered being infective. Degree of infectivity is elevated if the ssRNA enters into the cell via transfection. 5 ‘ UTR consists of trefoil foliage secondary construction which is called as ( IRES ) internal ribosome entry site. Both the terminals of genome are altered ; 5 ‘ terminal by little protein Vpg ( covalently attached ) and is composed of about 23 aminic acids. And the 3 ‘ terminal is modified by polyadenylation. The picorna viruses with the RNA molecules size ranges from 7.2-8.5kb in size deficiency a lipid envelope. These viruses can be inactivated by ionising radiation, phenol and methanal. After infection interlingual rendition is initiated with aid of ribosomes which leads to synthesis of polyprotein molecule ( weight 250, 000 )
Picornaviruss have been classified harmonizing 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
Rhinoviruss chiefly infect pharynx and nose unlike enteroviruses which chiefly infect enteral piece of land. Both the genera have indistinguishable morphology but they can be identified based on clinical, biophysical and epidemiological surveies.
Table1: Genre in Picivirdae household ( Ref: www.virology-microbiology-b.blogspot.com )
The nuclecapsid of a picorna virus has a 12 penton construction, one at each vertex of the virus. Surrounding each penton there are five other proteins. The virus has a protein composite which resembles a triskele construction on its each face. This is in bend in contact with six other proteins. The part between the pentons and triskele is called a canon, to with the cell surface binds in and conformational alterations takes topographic point to the viral proteins.
The mirid bug of these viruses consists of 60 boosters dumbly packed in an icosahedral agreement. Picornavirus has a nonenveloped construction. The 3-D construction of picornavirus was determined utilizing X-Ray crystallography. Each of the 60 boosters consists of 4 polypeptides, Etoposide ( VP ) 1, 2, 3 and 4. These polypeptides are derived from the cleavage of a larger protein. The chief maps of the mirid bug proteins are to protect the virus from environmental RNAses, host and cell tropism, presenting the viral RNA to the cell cytol by perforating the host cell and choice and packaging of viral RNA. VP1, VP2 and VP3 wrap themselves into a jelly form axial rotation or cuneus forms construction which is composed of ? barrel 8 stranded constructions. Capsid of picornavirus is formed by these 3 proteins unlike VP4 which is present below the shell. The mirid bug proteins of different works and animate being viruses portion similar creases.
Many picornaviruses contain depressions called as cavities or canons on the surface of mirid bug that plays function in fond regard to the surface of receptors. Depression or canon or cavity is non found in other types of picornaviruses. Foot and mouth disease virus and rhino virus make usage of enodcytosis ( receptor – mediated ) to come in the cell. Acidification caused in the endosomal cyst leads to conformational alteration within the mirid bug doing dislocation of fractional monetary units of mirid bugs and viral RNA release. In other types of picornaviruses such as poliovirus experience conformational change on the cell surface straight on attaching with the receptor.
Figure 1: shows the general reproduction form of Picornaviruss
( Ahlquist et al. , 2003 ) .
The continuance of a reproduction in picornavirus is 8-10 hours. The rhythm continuance may change depending on pH, temperature, cell types and figure of viral atoms that infect the cell. The general procedure of reproduction is that the virus comes in contact with the host cell. Enters the cell by agencies of endocytosis and the uncoating procedure happens. Then the viral RNA are transcribed and translated by cell enzymes and ribosomes. The cells productiveness is taken over by the virus and new virus atoms are produced by the cell. Within 30 proceedingss of the virus infection, the cellular protein synthesis declines about reaching nothing. This is called ‘SHUT OFF ‘ . The SHUT OFF is due to the cleavage of the cellular protein eIF-4G, a constituent of 220KD cap-binding composite ( CBC or CBP ) .
The picorna viruses attach themselves to the host cell by specific receptors. In the instance of infantile paralysis virus, a type of picorna virus the receptor protein is CD155 whose map 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 sing picornavirus reproduction came into image after conductivity of single-step growing curve experiments. Replication either occurs in cytol or in enucleated cells. The reproduction in this instance can non be inhibited by the action of actinomycin D. RNA dependent RNA polymerase produces ( – ) strands copying the genomic RNA A replicative intermediate composite which is multi stranded aid in RNA synthesis. ( – ) RNA acts as a templet for ( + ) strands. Two possible replicative theoretical accounts have been suggested after extended written text surveies ( in vitro ) .The entire figure of ( + ) strands synthesised exceeds the figure of ( – ) strands by ratio of 40:1. During first viral infection RNA of virus is formed in combination with membrane cysts. These cysts play function in placing the elements needed for sufficient RNA reproduction in the host cell. Picornavirus proteins 2C, 2B, and 3A aid in suppression of protein secernment every bit good as suppression of membrane trafficking via Golgi setup to plasma membrane. This leads to formation of cysts in the septic cell.
Fig2: Reproduction mechanism of Picornavirus ( Microbiology Bytes, 2007 ) .
The interaction between mirid bug and genome although is non really clear but still RNA is considered to be packaged into the pre formed capsids.The mirid bug is formed after the cleavage of precursor of P1 polyprotein into booster comprising of VP3, VP1 and VPO. They combine together therefore enveloping the genome. Viral genome is packaged in the mirid bug in the presence of substances such Na to call off ( – ) charge caused by phosphate group on RNA. Diameter of viral atom is 27-30 nm depending on desiccation grade.
Fig3: Diagram demoing the cleavage of polyprotein to organize mature virion
( Referee: hypertext transfer protocol: //cmbi.bjmu.edu.cn/www-learn/micro-ac-uk/335/Picornaviruses.html )
For case when we consider the binding of Rhinoviruss 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 cardinal portion of the penton surrounds a closed pore. The canon will be present where the VP1 Opposes VP2/3. At the floor of the canon, there is a narrow hole holla which a bigger infinite called pocket is present. The pocket contains ‘pocket factor ‘ . Another protein VP4 is present below VP1, 2 and 3 which can non be seen from outside. The ICAM-1 tantrums into the VP1/2/3 composite, which fits into the canon but this does non perforate into the pocket. When ICAM binds in the canon, a conformational alteration which tips back VP1 from Centre of the penton takes topographic point. This squeezes the pocket and perchance the binding displaces the pocket factor. When VP1 tips back, the pore opens as VP3 moves out of the manner. This allows VP4 to travel through the pore. The formation of a pore is besides helped by the fact that the VP1 amino end point is an amphipathic alpha spiral which flips out ICAM-1 binding.
Fig4: Diagram to demo interlingual rendition procedure in Picornavirus
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After the virus binds to the protein and enters the cell the procedure of uncoating takes topographic point. There is a possibility that the virus after adhering might acquire eluted once more. When that happens the virus composite go through the first measure of uncoating by losing the VP4 protein with the exclusion of not infective A atom. And when the virion binds to the membrane of the host, it forms a coated cavity construction and undergoes the above reactions and eventually the Ribonucleic acid from the shell is released into the host cytol and empty shells are expelled out.
The host cellular maps are influenced by viral proteolytic activities. The major peptidases involved are the 2A and 3A which includes Lpro, 2Apro, and 3Cpro. These are picorna virus encoded peptidases which are of import for viral polyprotein processing ( Strebel et al. , 1986, Joyoda et al. , 1986 ) . The viral peptidases non merely split viral polypeptides but besides inhibit assorted host mechanisms. The 3Cpro enters the karyon through its precursor 3CD ‘ or 3CD, which has a atomic localisation sequence ( NLS ) ( Sharma et al. , 2004 ) . The 3Cpro cleaves legion factors and regulators associated with cellular DNA-dependent RNA polymerase I, II and III such as TATA box adhering protein ( TBP ) , Octomers-binding proteins ( OCT-1 ) , written text activator p53, cyclic AMP-responsive component adhering protein ( CREB ) , histone H3 and DNA polymerase III ( Weidman et al. , 2001 ) . 3Cpro may besides be involved in virus-induced obstruction of host written text. 2Apro cleaves TBP but can non suppress cellular written text ( Yalamanchili et al. , 1997 ) . Numerous cytoskeleton-associated factors are cleaved by 3Cpro and 2Apro.
The 2Apro of enterovirus and rhinoviruses carryout the primary cleavage between the mirid bug and non-structural precursors on the several polyproteins. The enzyme cleaves at the C end point of VP1 and its ain N-terminus. The Lpro of FMDV in contrast cleaves between its ain C-terminus and N-terminus of VP4. Both the 2Apro and Lpro are responsible for specific proteolysis of a host cell protein, the eucaryotic induction factor ( eIF ) 4G ( Lloyd et al. , 1988 ) . The cleavage of eIF4G inhibits the cell to originate the protein synthesis on its ain messenger RNA. The viral protein synthesis is initiated internally by the ribosomes on the IRES ( Internal ribosome entry section ) within 5’UTR ( Jackson et al. , 1990 ) . The eIF4G cleavage does non happen at the same peptide bond. Lpro cleaves between residues Gly479 – Arg480 and 2Apro cleaves at Arg486 – Gly487. Though the cleavage takes topographic point at different bonds, the consequence of the cleavage is same.
A survey by Ryan et al. , ( 1997 ) , shows that 3Cpro is unlike the other two peptidases. The 3Cpro is responsible for a series of secondary cleavages ensuing in the processing of mirid bug and replicative proteins precursors. The 3Cpro mediated cleavages occur at Gln-Gly braces. The brace within the peptidase are non processed. The processing of the mirid bug protein precursors is really mediated by 3CDpro, non by 3Cpro. The autoprocessing of the 3CDpro to 3C and 3D is enhanced by pure 3AP protein add-on. This besides stimulates the processing of proteins 2BC.The ability of a poliovirus 3Cpro to procedure precursors was studied by building chimeral polyproteins in which the parts of the poliovirus complementary DNA encoding 3C was replaced by tantamount 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 mirid bug protein precursors was non processed by either HPV-14 or CB3 3Cpros.
The membrane changes in the septic cells are caused by viral protein 2B and its precursor 2C. The 2B and 2BC contain two hydrophobic parts, which are ? spiral spheres. This helps the protein to incorporate into the membrane of the host Golgi and ER composite which produces virus induced cysts and virporin composite. The accretion of 2B or 2BC protein leads to plasma membrane and dismantling of gogi composite.
The 3A protein is a membrane binding protein which inhibits cellular protein secernment and interceding presentation of membrane proteins during viral infection. The 3A protein look causes ER to Golgi trafficking. The protein trafficking by 3A is caused by the break of ADP-ribosylation factor ( Arf ) household, which is an of import constituent of the membrane secernment tract ( Belov et al. , 1997 ) .
The 3AB protein is a multifunctional protein with the 3A part of the protein associated with membrane cysts ( Jowner et al, 1996 ) . This interaction anchors the reproduction composite to virus induced cysts. The membrane associated 3AB protein binds straight to precursor 3CD on the RNA of poliovirus. This stimulates the activity of the 3CD and serves as an ground tackle for 3D polymerase in the RNA reproduction composite. The 3AB maps as a substrate for 3D polymerase in VPg uridylylation. The 3AB of the poliovirus was observed to demo other maps such as spiral stabilisation. This shows that 3AB has nucleic acerb chaperon activity in destabilising the secondary construction of RNA and enhances the hybridisation in complimentary nucleic acids in viral reproduction ( Destefano et al. 1999 ) .
The enteroviral and rhinoviral 3B proteins ( VPg ) are little 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 peptidase activity but no polymerase activity. The 3CD aids in viral RNA reproduction by circularisation of the viral genome via interacting with both 5 ‘ and 3 ‘ terminals of viral RNA ( Harris et al. , 1992 ) . PCBP1 and PCBP2 are KH spheres RNA binding proteins, which are involved in the metamorphosis of cellular messenger RNA ‘s in normal cells. The add-on of recombinant PCBP1 rescues viral RNA reproduction in PCB depleted infusions but does non deliver viral interlingual rendition ( Harris et al. , 1992 )
The 3D protein is one of the major constituents of the viral RNA reproduction composite. It is a RNA-dependent RNA polymerase. Elongation activity is observed in the purified poliovirus 3D polymerase ( Van Dyke. , 1980 ) . 3D polymerases can besides uridylylate VPg and utilize VPg-pUpU as a primer during viral RNA reproduction ( Paul et al. , 1998 ) .The polymerase-polymerase reaction of poliovirus has been observed in biochemical and crystal construction surveies. The polymerase oligomerization has been proposed to be responsible for efficient template use. The RNA secondary construction in the viral genome dramas of import functions in the reproduction of viral RNA. The Cis elements contain stem-loop I at the 5 ‘ end point of 5’UTR, 3’UTR and poly ( A ) tail at the 3 ‘ end point of enterovirus RNA. The circularisation of the polioviruses template between the 5 ‘ and 3 ‘ terminus of the viral genome is important during the induction of both positive and negative strand RNA reproduction ( Pata et al. , 1995 ) . The binding of 3CD and 3AB to 3’UTR does non depend on the interaction with host proteins and suffices for viral RNA reproduction. The 3 ‘ stem-loop I of the negative strand is the induction 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 function in interceding cross-talk between the 5 ‘ and 3 ‘ terminals of CVB3 RNA for viral RNA reproduction ( Pata et al. , 1995 ) .
Fig 5: Shows the sum-up of the Proteolytic activities taking topographic point in the reproduction of Picornavirus
( Journal of biomedical scientific discipline, 2009 ) .
The proteolytic activities taking topographic point inside the picorna virus is responsible for its reproduction. When a host is infected by the picorna virus the proteolytic activity of the host bit by bit reaches nothing. This is called SHUT OFF and this is caused fellow to the proteins synthesised by the picornavirus. The chief peptidases are the 2Apro, Lpro and 3C pro. The breakage of proteins leads to the procedure of reproduction of these viruses. The proteolytic activity of the host is disrupted by the cleavage of eIF-4G. The picornavirus will non retroflex if any one of these peptidases are non synthesised. Thus the modern drugs to bring around diseases caused by picornoviruses target these protein production tracts.