M~6A Modification Effect And Regulation Mechanism Of SINV Replication

Sinderbis virus(SINV)is a member of the genus Alphavirus in the family Togaviridae,belong to Martellivirales.The viral genome is single plus-stranded RNA,encoding non-structural polyprotein and structural polyprotein,non-structural polyprotein is cleaved into four different proteins(ns P1,ns P2,ns P3,and ns P4),the structural proteins include Capsid,E3,E2,6K and E1.SINV is a zoonotic virus distributed in Eurasia,Africa,Oceania et al.SINV can cause fever,rash and arthritis in humans.m~6A is the most the most abundant internal modifications of RNA and it is generally enriched near the stop codon and terminal exon.m~6A is involved in the regulation of various processes of RNA processing and metabolism,including m RNA transcription,splicing,nuclear output,localization,translation and stability,thus playing a role in various processes of regulating life activities.Several studies have shown that m~6A modification plays a crucial role in regulating viral replication.m~6A modification allows the virus to evade the innate immune response in addition to directly regulating viral replication by affecting the stability of RNA.The role of m~6A modification in alphavirus is still unclear.Recent studies have shown that m~6A modification can regulate the replication of CHIKV,but the mechanism is unknown.Autophagy is an internal homeostasis mechanism in eukaryotic cells,which can degrade cellular components against nutrient deficiency and play an important role in antagonizing intracellular pathogens.Autophagy can sense viral infection and has antiviral or pro-viral effects.Meanwhile,viruses promote viral proliferation by evolving strategies to evade or exploit autophagy pathways.For example,studies have shown that SINV can protect the central nervous system of mice from infection by degrading viral proteins through autophagy.Some viruses,such as coronaviruses,poliovirus,and foot-and-mouth disease virus(FMDV),use autophagosomes to replicate.At present,more and more studies indicate that there is mutual regulation between m~6A modification and autophagy.However,it is not clear whether m~6A modification interacts with autophagy to regulate viral replication.Research methods and Results:(1)m~6A immunoprecipitation high-throughput sequencing(Me RIP-Seq)was used to determine that there was significant m~6A enrichment in the nucleocapsid region of the SINV genome,suggesting that m~6A modification existed in the SINV genome.(2)Immunofluorescence technique was used to determine the subcellular co-localization of m~6A methyltransferase with SINV double-stranded RNA(ds RNA)and Capsid.The results showed that after SINV infection,viral nucleocapsid protein Capsid,glycoprotein E2 and viral ds RNA were co-located with m~6A methyltransferases METTL3 and METTL14 in cytoplasm.Meanwhile,the virus ds RNA co-located with the m~6A reading protein YTHDF1-3 in the cytoplasm.These results suggest the presence of m~6A modifications in SINV genome,which may be mediated by co-localization of the virus with methylase and reading protein in cytoplasm.(3)Real-time fluorescence quantitative technique and Western Blot were used to detect that SINV infection did not affect the m RNA levels of m~6A methyltransferase METTL3 and METTL14,as well as the protein levels of m~6A reading protein YTHDF1-3.This suggests that SINV infection does not directly regulate m~6A modification molecules in cells.(4)Real-time fluorescence quantitative technique,Western Blot and plaque assay were used to detect the relationship between 3-DAA,m~6A methylase and viral replication after SINV infection.After 3-DAA treatment,virus gene copy number,nucleocapsid protein expression and virus titer in cell culture supernatant were increased,indicating that SINV replication was promoted,but whether the specific mechanism depended on m~6A pathway was not clear.However,the virus gene copy number and nucleocapsid protein expression in SINV-infected METTL3 deficient cells did not change significantly,and the virus titer in cell culture supernatant was only affected in the early stage of infection,but basically had no effect in the late stage of infection,indicating that m~6A methylase METTL3 had basically no effect on the replication of SINV.(5)Further Western Blot and plaque assay were used to determine whether the m6A reader YTHDF1-3 affected SINV replication.The results showed that both YTHDF1 and YTHDF2 inhibited the exocytosis of virion,while YTHDF3 promoted the exocytosis of virion.It is suggested that YTHDF1-3 are involved in the regulation of SINV replication,but the specific mechanisms are different.YTHDF1 and YTHDF2 may inhibit viral replication by affecting m RNA stability,while the mechanism of YTHDF3 promoting viral secretion needs further study.(6)In order to determine whether YTHDF3 affects SINV secretion by regulating IFN pathway,q PCR was used to verify the expression of interferon stimulating gene(ISG)molecules such as IFN-β,Mx1,IFIT2 and BST2 in cells with YTHDF3 deletion after SINV infection.The results showed that YTHDF3 did not regulate SINV exocytosis through interferon pathway.RNA-Seq sequencing analysis showed that several autophagy related genes were changed in YTHDF3-deficient cells after SINV infection.q PCR and Western Blot were used to detect the levels of autophagy related molecules,The results showed that the expression levels of autophagy receptor SQSTM1,LC3-I and LC3-II were significantly decreased in YTHDF3-depleted cells,suggesting that YTHDF3 may affect the release of virions by blocking autophagy in host cells.(7)Immunofluorescence and transmission electron microscopy were used to observe autophagy in YTHDF3-deficient cells after SINV infection,and it was found that the number of autophagic lysosomes in YTHDF3-deficient cells increased significantly after SINV infection.Further Western Blot was used to detect that after SINV infection,The expression levels of SQSTM1 and LC3-II in YTHDF3-deficient cells were significantly decreased,indicating that complete autophagy had occurred,while YTHDF3supplementation could offset the changes in autophagy process caused by YTHDF3-deficient cells,suggesting that SINV could induce complete autophagy in YTHDF3-deficient cells.(8)The cells were treated with Bafilomycin A1 and chloroquine,which are late inhibitors of autophagy,to further study the mechanism of SINV inducing complete autophagy in YTHDF3-deficient cells.The mechanism of complete autophagy induced by SINV infection in YTHDF3 deficient cells was further studied by treating cells with advanced inhibitors of autophagy,Bafilomycin A1 and chloroquine.The results showed that the expression of SQSTM1,LC3-II and Capsid increased 3 hours after SINV infection treated with chloroquine and Bafilomycin A1.The results showed that YTHDF3 may regulate autophagy during SINV infection by affecting the fusion of autophagosome and lysosome.In conclusion,SINV has m~6A modification,which is related to methylase and reading protein.m~6A reading protein is involved in the regulation of SINV replication,among which YTHDF3 affects viral secretion and has nothing to do with IFN pathway.The mechanism may be that SINV exocytosis is affected by regulating autophagy process.