The Effect And Mechanism Of Capsid Protein VP0 Pivotal Amino Acid Sites On The Assembly And Penetration In EV71 Virus

Enterovirus 71?Enterovirus71,EV71?belongs to the family of Picornaviridae and is the main pathogen causing hand,foot and mouth disease?HFMD?in infants and young children.It can cause severe neurological diseases such as aseptic meningitis,encephalitis,and polio-like paralysis and severe can lead to death.The capsid proteins in Picornavirus are crucial for virus assembly and invasion.However,in EV71 virus,there are few reports about the effect of specific amino acid sites of capsid protein on virus function.Based on the study of the capsid proteins in other Picornavirus,it is known that the myristic acid site on VP0 affects the virus invasion and uncoating process;the VP0 cleavage site affects the assembly and maturation of the virus capsid.However,for EV71 virus,the location and function of these two types of sites have not been reported,so understanding their mechanism is crucial for the prevention of EV7 virus and the discovery of antiviral drugs.In this thesis,it is divided into two parts:the study of myristic acid binding site and the capsid protein VP0 cleavage site.Part ?:The effect of myristic acid binding site on EV71 virus penetrationMyristylation is the process of covalent attachment of the N-terminus of the protein to myristic acid.Some studies have reported that myristoylation of Picornavirus affects virus assembly and infectivity,and is an important amino acid site that affects the life cycle of the viruses.In EV71 virus,the site of myristoylation is the second glycine at the N-terminus of VP4Firstly,we constructed the EV71 myristoylation-deficient luciferase reporter pseudovirus?Pseudo-G2A?and myristoylation-deficient live virus?Live-G2A?full-length plasmids.By transfecting the plasmids into 239T cells,we can obtain the myristoylation-deficient pseudovirus and live virus.Western-blot detection of viral proteins and electron microscopy of the appearance of the virus revealed that the loss of myristoylation did not affect these aspects.Secondly,we infected 293S cells?Stable expression of EV71 receptor SCARB2?with the Pseudo-WT/G2A and Live-WT/G2A,respectively,and detected the intracellular luciferase protein expression level and viral RNA amount in infected cells.It is found that the luciferase protein decreased to 1/500 in the Pseudo-G2A compared with the pseudo-WT.More,the amount of viral RNA decreased to 1/50compared with the pseudo-WT after infection.In the live virus,the detection of cell CPE?Cytotoxic effect?and the western-blot of viral capsid proteins revealed that the CEP was not observed in the cells infected by Live-G2A,and the viral protein was not detected either.Blind passage of cells infected by Live-WT/G2A was carried out to measure the amount of viral RNA in every generation cells.By immunofluorescence analysis of infected cells,we found that the loss of myristoylation seriously blocked the replication of viral RNA and greatly inhibited the synthesis of viral progeny.In conclusion,the absence of myristoylation drastically decreased the infectivity of the virus.Next,in order to further elucidate the mechanism that the inhibition of viral RNA replication by lossing of myristoylation,we did the single and multiply growth curves of the virus respectively.The results show that the loss of myristoylation inhibited the viral genomic RNA replication;meanwhile,the content of viral negative-strand RNA is also severely limited.The mechanism was that the virus could not synthesize negative stranded RNA during the replication process,which affected the early step of viral infection.Finally,there are reports suggesting that myristoylation mediates rhinovirus?HRV?membrane penetration.We hypothesized that the role of myristoyl group is to interacting with the membrane structure in the cell to mediate the uncoating of genomic RNA during viral infection.In order to test this hypothesis,we constructed a myristoylation-deficient VP4-EGFP fusion expression plasmid?VP4-G2A-EGFP?,and confocal immunofluorescence observation after transfecting plasmids into 239T cells revealed that compared with the VP4-WT-EGFP aggregately distributed in specific organelles,the VP4-G2A-EGFP is basically dispersed in the cytoplasm.For more detailed explanation,we separated the cytoplasm and membrane structure from the cells transfected with the plasmid,respectively.Western-blot detected viral protein distribution of each component.It was found that most of the VP4-WT-EGFP proteins were distributed in the membrane structure,while the VP4-G2A-EGFP was mainly distributed in the cytoplasm.In addition,in order to directly investigate the membrane penetration function of myristoyl groups,we artificially synthesized EV71-VP4-WT and EV71-VP4-G2A polypeptides.Penetration experiments with liposomes containing luminescent compounds showed that the myristyl groups interact directly with the membrane.This interaction makes the membrane structure more penetrated,which may create conditions for virus uncoating and genome injection into the cytoplasm after endocytosis.In summary,we show that the myristyl group at the N-terminus of EV71 VP4directly interacts with the membrane structure,which may be a necessary condition for the endocytosis of virus capsid and genome injection into the cytoplasm.Therefore,the loss of myristoylation inhibits the synthesis of EV71 negative strand RNA and the replication of the viral genome,which seriously decreases the infectivity of the virus.But the loss of myristoylation does not affect the viral protein expression and the appearance of the virus particles.Part ?:Effect of VP0 cleavage site on EV71 virus assemblyThe cleavage of the capsid protein VP0 into VP4 and VP2 is a key step in Picoranvirus assembly.Viruses become infectious only after VP0 cleavage is completed.However,the cleavage site of EV71 VP0 and the action force to promote this behavior have not been determined.Previous studies have shown that mutations in amino acid residues on both sides of the VP0 cleavage site can lead to incomplete or noncomplete cleavage of the VP0,but different mutations at the cleavage site have not been studied in detail.First,we determined that the EV71 VP0 cleavage site was located at 69K/70S by mass spectrometry analysis of purified EV71 VLP and virus.Secondly,by construction,we obtained different mutant plasmids of VP0cleavage site.After transfecting the plasmid into 293T cells,the supernatant was infected with 293S cells,and the detection of the nucleic acid and protein levels of the infected cells revealed a single mutation in the 69K groups,including the T69S70 and P69S70,virus replication was not affected;single mutation 70S and double mutation groups,including K69A70,K69T70,T69P70 and F69K70,The virus replication was severely decreased,the cells did not show CPE,and no viral proteins were detected intracellularly.According to the above results,it was found that as long as the mutation of 70S,the virus could not be assembled,and the infectivity was lost.Therefore,this study determined that 70S is indispensable for virus assembly.Finally,we also conducted a preliminary study on the VP0 cleavage mechanism.The constructed plasmid,2A^pro and 3C^pro,were co-transfected with the VP0 plasmid.It was tested whether it had cleavage effect on VP0.By western-blot detection,it was found that 2A^pro and 3C^pro did not participate in the cleavage of VP0.In summary,this part of the study demonstrated that the VP0 cleavage site is located at 69K/70S.By detected viral nucleic acid and protein levels,it is proved that the 70S site has a greater influence on the assembly and infectivity of EV71 virus.Mechanism studies confirmed that 2A^pro and 3C^pro are not involved in the cleavage of VP0.This study has demonstrated that the myristic acid binding site and VP0 cleavage site on the capsid protein VP0 affect the assembly,penetration,and infectivity of the EV71 virus.It is speculated that the loss of myristoylation affects the interaction between the virus and the uncoating related membrane during the virus invasion,making it impossible for the virus to complete the uncoating,the viral negative-strand RNA can not be synthesized,and then the viral RNA replication is inhibited,eventually leading to the reduction of viral infection.VP0 cleavage site locates at K69and S70,and the S70 amino acid site is more important for virus assembly and maturation.2A^pro and 3C^pro of EV71 virus do not participate in the cleavage process of VP0.