Molecular Evolution Of Enterovirus 71 And The Role Of Hsa-let-7c-5p In Its Replication

Backgroud:Human enterovirus 71(EV71)causes severe hand,foot and mouse disease(HFMD)in young children,accompanied by neurological complications.Large-scale and even serious HFMD outbreaks have been caused by EV71 in the past several decades.However,it is unclear whether VP4-based genetic classification is consistent with that based on VP1 gene in the molecular epidemiology study of EV71,and no comprehensive study is performed to explore comparative analysis of genotyping based on these two genes.Moreover,little is known about the evolution of EV71 VP4 gene.In addition,the phylogeographical information on EV71 remains unclear.Thus,it is of important significance for study of molecular evolution and phylogeography of EV71 to dig the biological information of VP4 gene from global EV71 for the decades,which may also provide a new kind of strategy and measure for surveillance,prevention and control of EV71 infection-caused HFMD epidemics.At present,no specific drug is available for clinical therapy for EV71 infection-caused HFMD.MicroRNA(miRNA),as a kind of nucleoside analogues,is of potential value for genetic therapy of diseases.During the interaction between EV71 and the host,the virus subverts host cell machinery for its own replication.The roles of miRNA hsa-let-7c-5p in mediating virus-host interactions through the regulation of host pathways have not been previously investigated.Moreover,the regulation of hsa-let-7c-5p in EV71 replication has not yet been reported.Therefore,study on hsa-let-7c-5p-mediated interplay between EV71 and the host may contribute to understanding the viral pathogenesis and providing new targets for future therapy.Objective:(1)To study the phylogenetic classification of EV71 based on VP4 gene and profoundly investigate the molecular evolution and phylogeography of EV71;(2)To study the mechanism by which hsa-let-7c-5p promotes EV71 replication through viral subversion of mitogen-activated protein kinase kinase kinase kinase 4/c-Jun NH2-terminal kinase(MAP4K4/JNK)axis and preliminarily investigate the mechanism by which JNK affects EV71 replication via autophagy.Methods:(1)The phylogenetic trees were constructed for EV71 genotyping using Neighbor-Joining method in MEGA 5 software,and the divergence of VP4 nucleotide sequences and the similarity of the deduced amino acid sequences within and between genotypes were further analyzed using the DNAstar-MegAlign program.The divergent time and evolutionary rates were inferred using VP4-based Bayesian phylogenetic method in BEAST 2 software.Polymorphism analyses of VP4 genes of EV71 population were performed with DnaSP software.Selective pressure on VP4 gene at amino acid level was estimated using four different codon-based maximum likelihood methods,which were available at the Datamonkey online version of the Hy-Phy package.Population dynamics of genogroup B and C viruses were analyzed with coalescent-based BSP method implemented in BEAST 2 software.(2)The spatio-temporial distribution of each genotype of EV71 was exhibited using leaflet program.Phylogeography of EV71 genogroups B and C were investigated using the discrete phylogeographical model implemented in BEAST 2 software,and the pMRCA and the possible transmitted routes of these two genogroups viruses were inferred.(3)During the interaction between EV71 and rabdomyosarcoma(RD)cells,the alteration in miRNA hsa-let-7c-5p expression after EV71 infection was detected using stem-loop qRT-PCR.The impacts of hsa-let-7c-5p mimic,hsa-let-7c-5p inhibitor and MAP4K4 knockdown on EV71 replication were identified using plaque assay,qRT-PCR,Western blotting and MTT assay.Targeting on MAP4K4 by hsa-let-7c-5p was estimated using Luciferase assay,qRT-PCR and Western blotting.The effects of silencing MAP4K4,hsa-let-7c-5p mimic and inhibitor on JNK and NF-?B signaling pathways were detected using Western blotting.The impacts of EV71 infection and UV-inactivated viral infection on JNK signaling pathway and the role of JNK inhibitor SP600125 in viral replication were identified using plaque assay,qRT-PCR or Western blotting.(4)The impacts of EV71 infection and UV-inactivated viral infection on autophagy-related proteins LC3 and P62 expression were detected using Western blotting.The impact of SP600125 on LC3 and P62 expression was detected using Western blotting or qRT-PCR.After RD cells were treated with autophagolysosomal inhibitor chloroquine,the impacts of EV71 infection on autophagolysosomal formation and chloroquine on viral structural protein VP1 expression were detected using Western blotting.Results:(1)Genetic classification of EV71 was divided into 3 genogroups(A,B and C)and 11 genotypes(A,B1 to B5 and C1 to C5)by VP4 gene,and genotype C4 was further divided into 3 subgenotypes(C4a1,C4a2 and C4b).Moreover,the genotyping based on VP4 phylogeny was kept consistent with that based on VP1 phylogeny.The origin of time for EV71 could be dated to mid-20 century,and the evolutionary rate of genogroup C was estimated to be slightly higher than that of genogroup B.And the divergent time of each genotype in these two genogroups was estimated to be earlier for 1-7 years than its first detection.The molecular evolution of VP4 gene followed the neutral model,and the VP4 coding sequence was under a strong purifying selection.Moreover,no amino acid site was under positive selection pressure.The homology of the deduced amino acid sequences of VP4 reached 91.3-100%.The fluctuations in genetic diversity of VP4 gene of genogroup B were not obvious compared to that of genogroup C,and the alterations in genetic diversity of this gene corresponded to the period when outbreaks or epidemics of HFMD associated with EV71 infection occurred.(2)EV71 mainly circulated in North America,Europe and the Asia and Pacific region.The geographical origins of EV71 genogroups B and C were estimated to occur in Netherlands from 1964.4 to 1965.2 and Japan from 1977.6 to 1981.4 with high certainty,respectively.A total of 9 and 13 statistically supported epidemiological links were inferred across the twelve geographical regions for the genogroup B viruses and fourteen geographical regions for genogroup C viruses,respectively.(3)Mature hsa-let-7c-5p expression was upregulated during EV71 infection in RD cells.The hsa-let-7c-5p mimic promoted replication of the virus,and the hsa-let-7c-5p inhibitor suppressed viral replication.Hsa-let-7c-5p targeted MAP4K4 and inhibited its expression.Downregulation of MAP4K4 expression led to an increase in EV71 replication.In addition,MAP4K4 knockdown or transfection with the hsa-let-7c-5p mimic led to activation of the JNK signaling pathway,whereas the hsa-let-7c-5p inhibitor inhibited activation of this pathway.EV71-induced JNK signaling pathway activation further facilitated viral replication.(4)EV71 replication induced autophay of RD cells.Blocking JNK signaling pathway suppressed EV71-induced autophagy.Chloroquine blocked EV71-triggered autophagolysosomal formation and suppressed viral structural protein VP1 expression.Conclusions:(1)VP4 gene could be applicable for EV71 genotyping,which may act as a marker for the molecular surveillance of EV71 epidemics and a potential target for antivial drugs.In addition,the potential transmitted routes of EV71 could be explored within or among North America,Europe and the Asia-Pacific region.(2)Hsa-let-7c-5p,upregulated by EV71 infection,facilitated EV71 replication by inhibiting MAP4K4 expression,which might be related to viral subversion of MAP4K4/JNK axis.In addition,the JNK signaling pathway may promote EV71 replication through triggering autophagy.