Effects Of Key Amino Acids Mutation Of Enterovirus 71 2A Proteinase On Viral Virulence

Enterovirus 71(EV71)belongs to the family of single-stranded,positive-strand RNA viruses ofPicornavirida.EV71 infections result in a myriad of diseases from mild hand,foot,and mouth disease(HFMD)to severe neurological complications that can be fatal in infants under 5 years old.The pathogenic mechanism of EV71 is unclear.Previous study suggested that there are multiple virulence candidate sites in the genome of EV71 and EV71 2A proteinase plays important role in viral replication and virulence.However,the accurate localization of virulence sites on 2APro needs further study.In this study,there are three site-directed mutants were constructed by using reverse genetics method to explore the function of 2Apro in the viral replication and virulence,this provides a theoretical basis for virulenced determination of EV71 and anti-EV71 drug development.Objective1.Comparing the amino acids sequence of 2A proteinase of two virulence phenotype strains of EV71,mutating the amino acids in virulent strain 2A to that of the attenuated strain,and constructing and rescuing site-directed mutants.2.To compare the differences in replication capacity,cell injury and autophagy between the mutated and virulent strains,and explore the roles of virulence candidate sites on 2Apro in the pathogenesis of EV71.Methods1.Using the overlapping PCR technology to obtain the mutated fragment of 2A,and then,the mutated fragment was swapped into the full length gene of SDLY107 to obtain site-directed mutants of EV71.Then the in vitro,synthesized RNA transcripts were then transfecting into RD cells to produce the rescued virus.The site-directed mutants was identified by PCR,indirect immunofluorescence assay(IFA),and verified by sequencing.CCID50 and plaque test were used to measure the virus titer.2.The RD cells,U87 cells and SH-SY5Y cells were used to detect the differences between site-directed mutants and their parental strains in replication capacity,pathogenicity and the induction of autophagy.Results1.There are three amino acids differences between 2Apro of EV71 different clinical phenotype strains,which are Y64H(full-coding region Y190H),M68R(full-coding region M203R),and D85E(full-coding region D255E).2.Three site-directed mutants were successfully constructed and rescued,and their virus titers were detected.3.The results of qRT-PCR showed that there was almost no difference in the replication capacity between site-directed mutants and parental strains in RD cells and U87 cells,but the virulent strain SDLY107 reached the peak of replication earlier.However,site-directed mutants lost replication capacity,while virulent strain SDLY107 and attenuated strain SDLY1 had the ability to replicate in SH-SY5Y cells.4.LDH assay and CCK8 assay showed that the cell injury of site-directed mutants were weaker than the virulent strain SDLY107 in RD cells,U87 cells and SH-SY5Y cells(P<0.05).5.Western Blot assay and immunofluorescence experiments showed that cells were infected with EV71 strains could trigger autophagy,and the ability of the EV71 strains to induce autophagy and the autophagic degradation mechanism were different in different cells.Conclusions1.The replication capacity of the three site-directed mutants were significantly different from that of their parental strains in nerve cells,indicating that the 64th amino acid(Y?H),the 68th amino acid(M?R)and the 85th amino acid(D?E)of 2Apro may be the key site affecting the replication of EV71 in nerve cells and is closely related to the neurotoxicity of EV71.2.The level of virus replication was reduced and the signal of the key molecules of autophagy was weakened after the site-directed mutants infected the nerve cells.This phenomenon indicates that the process of autophagy may be related to their replication capacity.