The Biological Function Of M114V Mutation In The NS5 Protein Of Zika Virus

Zikv virus(Zikv)is a newly re-merging arbovirus which belongs to flavivirus genus of the Flaviviriade family.Zika virus was first discovered and isolated from a febrile rhesus macaquein the Zika Forest of Uganda in 1947.Besides Zikv,many flavivirus are human pathogens,including Japanese encephalitis virus(JEV),yellow fever virus(YFV),dengue virus(DENV)and so on.Until 2007,without causing detected severe diseases or large out-breaks,ZIKV had silently circulated in many parts of Africa and Asia with fewer than 20 documented human infections.However,during the recent epidemics in Brazil,Venezulela and other regions of latin America,ZIKV infection has caused devastating severe diseases,including congenital malformations in the fetus of infected pregnant women(microcephaly and fetal demise)and Guillain-Barré syndrome in adults,which has raised global attention.There were only few ZIKV infections reported with mild clinical symptoms,and it had not been valued until recent years.Most cases have no symptoms,even present they are usually mild and symptoms generally last less than seven days.The symptoms can resemble dengue fever including fever,red eyes,joint pain,a maculopapular rash and so on.The occurance of serious neurological symptoms like microcephaly has exceeded our understanding of flavivirus infection and it is urgent to clarify the pathogenic mechanism of ZIKV.Phylogenetic analysis revealed that ZIKV evolved long ago into African and Asian lineages.Strains from the Asian lineage are responsible for the recent large-scale epidemics in the Americas in 2016.The recent emergence of ZIKV could be driven by a number of potential mechanisms,including the acquisition of genetic changes that increase neurovirulence and ability to infect in humans and mosquitoes.Indeed,a single amino acid substitution in ZIKV PrM protein was recent shown to lead to more significant microcephaly in mouse fetus and infectivity in neural progenitor cells.This may reveal why ZIKV causes microcephaly.Another mutation recently reported to increase viral infectivity for Aedes aegypti mosquitoes.Here we report that the 114 th amino acid on the NS5 non-structural protein of the current pandemic strain of ZIKV is highly conserved before 2014,then it was mutated from methionine(M)to proline(V).Further studies have found this single amino acid substitution enhance the ability of ZIKV to cross the blood-fence barrier and reduce the ability of the virus to interferon-antagonize.Our results demonstrate that ZIKV has accumulated a series of adaptive mutations during the epidemic virus,which may lead to a pandemic outbreak.My research is divided into two parts.First section: Interplay between different isolates of Zika virus and type I interferonIn order to compare Zika virus' s ensitivity to type I interferon,We selected several strains of Zika virus isolated from different eras and determined their sensitivity to IFN-?.The results showed that the early Zika strain had stronger IFN-? sensitivity than the current Zika strain.VEN/2016 has the least sensitive to IFN-?,but CAM/2010 has the most sensitive to IFN-?.The two viruses were further compared by experiments in vivo.The results showed that VEN/2016 was significantly more neurovirulent than CAM/2010;VEN/2016 caused more serious viremia than CAM/2010 in 129 mice;but there was no significant difference in viremia in A129 mice.It is speculated that the cause of this difference is related to type I interferon.Further experimental results show that VEN/2016 can prevent STAT2 from entering the nucleus,while CAM/2010 cannot.Under the effect of IFN-?,the expression of E protein in VEN/2016 was higher than that in CAM/2010.The above results indicate that the early isolates of Zika virus have a stronger susceptibility to interferon than the current epidemic strains of Zika virus,and it also lays a foundation for subsequent investigation of its pathogenic mechanism.Second section: The evaluation of the recombinant mutant virus M114 V and the parental virusThe largest protein encoded by Zika virus the non-structural protein NS5.The 2'O methylation of the N-terminal methyltransferase(MTase)allows the virus to escape the monitoring of innate immunity.In addition,studies have shown that Zika virus NS5 protein can degrade human STAT2 through the proteasome,hindering the production of downstream antiviral proteins.In order to explain the mechanism of sensitivities to IFN-? among different Zika virus,we systematically analyzed the amino acid sequences of different Zika virus,including VEN/2016 and CAM/2010.We found that the 114 th amino acid of Zika virus NS5 protein was highly conserved before 2015,and it evolved from methionine(M)to proline(V)after 2015.It is speculated that the mutation of this site affects the pathogenicity of Zika virus.We introduced the amino acid difference site of NS5 protein into the CAM/2010 infectious clone by reverse genetics technology,and obtained the recombinant mutant virus M114 V by means of transfected cells.The mutant virus and parental virus(WT)were further compared on different models.We found there were no significant differences in the plaque morphology,growth curves on different cell lines,and E protein expression of the two viruses.WT caused more serious viremia and organ tropism than M114 V in immune-competent 129 mice;but there was no significant difference in viremia and organ tropism in A129 mice.It is speculated that the cause of this difference is related to type I interferon.The results in vitro experiments showed that WT has less sensitive to IFN-? than M114 V.The results of downstream antiviral protein expression of interferon showed that WT has more potent than M114 V in antagonizing IFN-?.The results of pregnant mice and in vitro experiments further showed that M114 V has stronger replication ability in pregnant mouse placenta and neural cell lines than WT.Overall,compared with WT,M114 V has weaker virulence in the periphery,but has stronger virulence in nerve and placental cells.In summary,the present study found that the current and early Zika virushave different susceptibility to IFN-?,and we analyze the phenomenon by bioinformatics: the amino acid at position 114 of NS5 is methionine.The replacement of M to V may lead to changes in viral virulence and pathogenicity.In vitro and in vivo models,we found that the change of amino acid at position 114 of the non-structural protein NS5 affects the pathogenicity of Zika virus.This study is of great significance for the deep understanding of the outbreak and virulence evolution of Zika virus.It also laid the foundation for the subsequent development of Zika virus drugs and vaccines.