The Transcriptome Analysis Of Host Cells Infected With SFTSV And The Mechanism Of SFTSV Induced Cell Autophagy

Severe fever with thrombocytopenia syndrome virus(SFTSV),a novel bunyavirus,was first discovered in China in 2009,and subsequently in South Korea,Japan,Vietnam,Thailand,Pakistan have also been reported.SFTSV is a tick-borne virus,causing severe fever with thrombocytopenia syndrome(SFTS)with a fatality rate of 30%,posing a serious threat to human health.At present,the occurrence and prevalence of SFTS are on the rise.However,there are no effective vaccines or therapeutics for SFTSV treatment,so in-depth study of the interaction between SFTSV and the host is of great significance for the vaccine development and antiviral treatment of SFTSV.A large number of studies have shown that viral infection can directly reshape the expression of host cell genes,including m RNA and lnc RNA.However,transcriptome changes in THP-1 cells induced by SFTSV infection remain unclear.Therefore,in this study,RNA-seq technology was used to analyze the changes of m RNAs and lnc RNAs expression profiles in THP-1 macrophages infected with SFTSV for 24 h and 48 h.The results showed that after 24 h of SFTSV infection,720 m RNA and 79 lnc RNA were up-regulated and 590 m RNA and 12 lnc RNA were down-regulated.After 48 h of infection,648 m RNA and 73 lnc RNA were up-regulated,and 973 m RNA and 23 lnc RNA were down-regulated.Among them,577 m RNA and 31 lnc RNA were differentially expressed at 24 h and 48 h after SFTSV infection.GO enrichment analysis results showed that the biological processes of the differentially expressed m RNAs were mainly involved in response to cytokine and immune system process,while the biological processes of the differentially expressed lnc RNAs were mainly involved in defense response,response to cytokine and metabolic process.KEGG pathway enrichment results showed that the differentially expressed m RNA was mainly involved in toll-like receptor signaling pathway,TNF signaling pathway,NF-κB signaling pathway,JAK-STAT signaling pathway,systemic lupus erythematosus signaling pathway and alcoholism signaling pathway,and the differentially expressed lnc RNA was mainly involved in TNF signaling pathway,cytokine-cytokine receptor interaction signaling pathway,systemic lupus erythematosus signaling pathway and alcoholism signaling pathway.Then,7 core hub m RNAs(IL6,IL10,CXCL10,UBA52,TNF and SRC,CDK1)and 2 hub lnc RNAs(XLOC＿083,027 and XLOC＿113,317)were screened by analyzing differentially expressed m RNAs and lnc RNAs.Since both NRIR and MIR3142 HG,two known lnc RNAs,were up-regulated at 24 h and 48 h after infection,we validated their roles in SFTSV infection,and found that NRIR negatively regulated type I interferon response during SFTSV infection,while MIR3142 HG positively regulated inflammatory response.Subsequently,transcription factors enrichment and alternative splicing of differentially expressed m RNAs were analyzed,and we found that transcription factors RELA and IRF1 positively regulated CXCL10 expression during infection.SFTSV infection could cause significant changes in alternative splicing of host cells.During SFTSV infection,skipped exon events accounted for the majority of the alternative splicing events,and two splicing factors,SRSF3 and TRA2 B,both had reduced skipped exon events,leading to the generation of non-functional transcripts.The results of this part of the study will provide important theoretical support for the study of the interaction between SFTSV and the host,and provide new targets and directions for the vaccine development and treatment of SFTSV.Autophagy is an intracellular stress protection mechanism that maintains cell homeostasis and balance by degrading intracellular aging organelles and invading pathogenic microorganisms.Current studies demonstrate that autophagy can directly regulate the process of viral infection.However,the relationship of SFTSV and autophagy remains largely unknown.Therefore,we chose autophagy,which plays a key role in the process of multiple viral infections,as our research direction.In our study,Western blot,immunofluorescence and transmission electron microscopy were carried out to detect the transformation from LC3-I to LC3-II,the formation of endogenous LC3 puncta and the observation of autophagy ultrastructure respectively in SFTSV infected Vero and He La cells.Our results demonstrated that SFTSV infection could induce autophagy in Vero and He La cells.Since autophagy is a dynamic process,the occurrence of autophagy may induce the formation of autophagosome,or inhibit the fusion of autophagosome and lysosome.Therefore,we further detected the expression level of autophagy substrate SQSTM1/SQSTM1,and found that the expression of SQSTM1 was increased during SFTSV infection,revealing that SFTSV might induce incomplete autophagy.However,we observed that the autophagy structure under SFTSV infection was monolayer structure by transmission electron microscope,revealing that such monolayer structure might be autolysosome.Therefore,autophagy turnover was detected by using the autophagy inhibitors chloroquine and bafilomycin A1,GFP-m Cherry-LC3 was detected by immunofluorescence,and the co-location of LC3 and LAMP1 was detected.The results showed that SFTSV infection could induce the fusion of autophagosome and lysosome and induce complete autophagy.To further clarify the mechanism of host autophagy induced by SFTSV infection,Western blot was used to detect the expression of a series of autophagy-related proteins and MTOR activity.The results showed that SFTSV infection could promote the expression of ULK1,BECN1,ATG101 and ATG5 and inhibit the activity of MTOR,indicating that SFTSV infection could induce autophagy by inhibiting the activity of MTOR and inducing the expression of ULK1,BECN1,ATG101 and ATG5.To further verify whether SFTSV-induced autophagy is dependent on traditional autophagy,RB1CC1,BECN1,ATG5,ATG7,ATG16L1 knockout cells were used to detect he transformation from LC3-I to LC3-II type and the formation of endogenous LC3 puncta by immunofluorescence and Western blot respectively under SFTSV infection.And we found that SFTSV-induced autophagy was traditional autophagy dependent on the RB1CC1-BECN1-ATG5 axis.In order to explore the role of autophagy in SFTSV replication,we used autophagy-related gene knockout cells infected with SFTSV to observe viral replication.The results showed that SFTSV replication was severely inhibited in these autophagy-related gene knockout cells.We then treated He La cells with the autophagy inhibitor 3-MA under SFTSV infection and found that SFTSV replication was also inhibited.These results indicated that SFTSV replication depends on autophagy.Since we found that SFTSV replication was greatly weakened when autophagy was inhibited,we speculated that SFTSV might replicate in autophagosomes.We detected the proteins carried by SFTSV virus particles purified by PEG8000 by Western blot.LC3-II,BECN1,PIK3C3,ATG14,ATG5,ATG7,ATG12,ATG16L1,LMAN1,a GOLGA2 and TGOLN2 were found in purified SFTSV virus particles.We also found that both SFTSV NP and Gn were co-localized with LC3 by immunofluorescence.Then,we found that SFTSV existed in LC3-positive autophagy structure by immunoelectron microscopy,and immunofluorescence showed that SFTSV Gn was co-localized with LMAN1,GOLGA2 and TGOLN2 respectively.These results indicated that SFTSV was replicated and assembled in endoplasmic reticulum-Golgi intermediate compartment and Golgi membrane-derived autophagosomes.Since we observed that SFTSV particles was released from autophagic vehicles,and we hypothesized that SFTSV particles might be released by autolysosomes.STX17 and VAMP7 are critical proteins involved in the fusion of autophagosome with lysosome.We used STX17 or VAMP7 knockout He La cells infected with SFTSV and found that inhibition of the fusion of autophagosome and lysosome reduced the release of SFTSV virus.Then,we found that SFTSV NP and Gn co-localized with LAMP1 and SFTSV existed in the double positive autophagy structure of LC3 and LAMP1 by immunoelectron microscopy,indicating that autolysosome was exploited by SFTSV for exocytosis.These findings further expand our understanding of the interaction between SFTSV and the host,and provide new strategies for the treatment of SFTSV.