Effects Of SFTSV Gn And Its Mutation Of Amino Acid 323 On Autophagy And Viral Proliferation

Sever fever with thrombocytopenia syndrome(SFTS)is a zoonotic disease caused by severe fever with thrombocytopenia syndrome virus(SFTSV)with high fever,leukopenia and thrombocytopenia as the main symptoms,its case fatality rate can up to 27%.However,there are currently no effective antiviral drugs or vaccines available.Once break out,it will cause major public health emergency with economic burden and mortality.SFTSV Gn plays a key role in mediating viral entry into cells,viral assembly and viral particle formation.We found the 323rd amino acid of Gn may play an important role in virulence.Therefore,we aimed to further identify key amino acid sites and verify their biological functions by comparing the sequences in GenBank and constructing eukaryotic expression vectors that can express Gn and Gn(I323V)respectively.In addition,the interaction between SFTSV and autophagy was studied,and the I323V mutation of SFTSV Gn had significant effects on viral replication,autophagy level and cell survival were explored,which provide a theoretical basis for the structure and function of Gn and development of antiviral drugs.Objective1.The key amino acid sites that may affect SFTSV virulence was screened out.2.The interaction between SFTSV,Gn and autophagy was explored.3.The effect of SFTSV Gn key amino acid mutation(I323V)on cell activity and viral replication was speculated.Furthermore,the possible mechanism by detecting the level of autophagy related proteins was explored.Methods1.Screening amino acid sites:Based on the amino acid mutation sites screened in the previous study,the sequences of SFTSV M fragments in GenBank was compared to further determine the key amino acid sites that may affect the virulence of SFTSV.2.Protein expression:Eukaryotic expression vectors that can efficiently express the target protein was constructed by overlap extension PCR and homologous recombinant cloning.Besides,BSR-T7/5 cells were transfected by liposome method to achieve eukaryotic expression.3.Cell viability detection:Detected the ability of protein expression and/or SFTSV infection to cause cell-damaging by CCK-8 experiment.4.Virus replication detection:Viral titers at different times were analyzed by qRT-PCR,and the dynamic curve of virus replication was drawn after SFTSV infection and/or express Gn.5.Detection of autophagy level:the expression of autophagy related proteins was detected by qRT-PCR and Western blot.Results1.The V323I mutation of SFTSV Gn may be an important amino acid site affecting virulence,which also plays a major role in the genetic evolution of SFTSV.2.The eukaryotic expression vectors pcDNA3.1(+)-Gn and pcDNA3.1(+)-Gn(I323V)were successfully constructed using double enzyme digestion and sequencing analysis.3.Indirect immunofluorescence and Western blot results showed that the expression efficiency of Gn was significantly higher than that of Gn(I323V).4.Western blot and mCherry-GFP-LC3 double fluorescence indicator system showed that SFTSV infection induced complete autophagy in Vero and BSR-T7/5 cells,and the status of autophagy flux can modulate SFTSV replication.5.Gn and Gn(I323V)caused varying degrees of autophagy and cell damage,and they can enhance different level of autophagy and cell damage induced by SFTSV.Moreover,overexpression of Gn had no significant effect on viral replication,but the virus proliferation rate was significantly decreased after the overexpression of Gn(I323V).Conclusions1.Complete autophagy is indeed induced under SFTSV infection and SFTSV replication is regulated by autophagy flux.2.SFTSV Gn promotes autophagy flux and further enhance the level of autophagy induced by SFTSV.3.SFTSV Gn may affect cell activity and viral replication by regulating autophagy flux,and the mutation of 1323V has a significant effect on host cells and viral replication.