Screening And Function Analysis Of Intracellular Interaction Protein For Snakehead Fish Vesiculovirus Glycoprotein

In 2014,our laboratories isolated a novel rhabdovirus,Snakehead Vesiculovirus(SHVV),from a diseased hybrid snakehead.It belongs to the genus Perhabdovirus of Rhabdoviridae,and causes serious harm to the aquaculture of snakehead in china.At present,there are no good prevention and treatment measures for this discovered pathogen.The study of the interaction mechanism between the virus and the host and the immune control of the host play an important guiding role in the prevention and control of the virus.Preliminary studies found that streaky snakehead cells(SSN-1)produced autophagy after infection with SHVV,and proved that the occurrence of autophagy can inhibit the replication of SHVV virus,and this autophagy is activated by SHVV virus G protein.On this basis,this topic explores which autophagy-related proteins interact with G protein in cells during SHVV infection and the corresponding functions of the interacting proteins,revealing that after SHVV-infected cells,G protein interacts in cells,and the proteins that induce autophagy are analyzed to analyze the relevant functions of interacting proteins in the process of virus infection of cells.The results of the study will provide a scientific basis for elucidating the cellular defense mechanism triggered by SHVV infection,and also establish a theoretical basis for the development of effective drugs and vaccines for the virus.The main findings are as follows:1.Transcriptome analysis of CCO cells overexpressing SHVV G protein To investigate the effect of SHVV G protein on CCO cells,by means of transcriptome sequencing and bioinformatics analysis,the changes of gene expression levels in CCO cell lines after overexpressing pc DNA4.0-G for 24 h were analyzed at the transcriptome level,and a total of 905 Differentially expressed genes(DEGs),including 502 down-regulated genes and 403 up-regulated genes.Through Gene Ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGGs)enrichment analysis,it was found that the RNA levels of autophagy-related genes,such as ATG family and Beclin-1,were significantly up-regulated.The results showed that SHVV G protein could significantly stimulate the transcription of autophagy genes in cells.2.Screening of SHVV G protein interacting proteins by Co-IP combined with mass spectrometryTo initially screen and identify SHVV G protein intracellular interaction proteins,co-immunoprecipitation(Co-IP)was performed on SHVV-infected CCO cells using rabbit polyanti-SHVV-G antibody.Host-interacting proteins of SHVV G protein were screened by LCMS/MS mass spectrometry,and bioinformatics analysis was performed,and Co-IP was used for identification.Twenty-two high-scoring proteins were screened by mass spectrometry,and 3 candidate interacting proteins related to autophagy were selected.Co-immunoprecipitation and subcellular localization were performed to verify the interaction between SHVV G protein and VDAC2.To further explore the effect of SHVV G protein on VDAC2,firstly,CCO cells were infected with SHVV at MOI=0.1,1 and 10 for 24 h;then,SHVV(MOI=0.1)and CCO cells transfected with pc DNA4.0-G were respectively infected 12,24 and 36 h,the expression levels of VDAC2 m RNA and protein were detected,and it was found that both m RNA and protein levels of VDAC2 were significantly increased.The above results indicate that VDAC2 protein is closely related to SHVV infection,and SHVV G protein can promote the transcription and translation of VDAC2 m RNA.3.Study on the function of VDAC2 and the occurrence of autophagy and virus replication after SHVV infectionAfter overexpression of VDAC2,SHVV infection could significantly up-regulate Beclin-1 to induce autophagy.After knockdown and inhibition of VDAC2,SHVV infection could inhibited the expression of Beclin-1 and inhibited autophagy.In addition,co-IP and indirect immunofluorescence experiments showed that VDAC2 could interact with Beclin-1,indicating that VDAC2 could regulate autophagy through Beclin-1 mediated autophagy pathway.During SHVV replication,overexpression of VDAC2 significantly reduced G protein m RNA and expression levels,while VDAC2 knockdown significantly increased G protein m RNA and expression levels.These results suggest that VDAC2 induces autophagy through the Beclin-1-mediated autophagy pathway,and cells can use this autophagy to inhibit viral replication.In summary,this study screened and found that VDAC2 is an intracellular autophagy-related protein that interacts with SHVV G protein by transcriptome and LCMS/MS mass spectrometry techniques.It was demonstrated that VDAC2 can induce autophagy by upregulating Beclin-1,and cells can use this autophagy to inhibit virus replication.This discovery revealed for the first time the intracellular interacting proteins and their related functions in the process of rhabdovirus-induced autophagy,and provided a new reference for studying the relationship between autophagy and viruses.At the same time,this study shows that VDAC2 plays an important role in the process of antiviral immunity in lower vertebrates,and can be a target for preventing related diseases and developing antiviral drugs.