The Effects Of EIF3i On VSV Replication In Nerve Cell And The Molecular Mechanism

Vesicular stomatitis is a zoonotic caused by Vesicular stomatitis virus(VSV),that can infect Cattle,horses,pigs and many other animals and person.Although no cases of VSV infection have been reported in China,studies on the pathogenic ecology of bat carrying virus in yunnan province by domestic scholars have found that bats are carriers of VSV,suggesting that the occurrence of this disease has potential risks.In recent years,peste des petits ruminants(PPR)and African swine fever(ASF),two foreign diseases have been introduced into China,causing great economic damage to animal husbandry.Therefore,it is of great scientific significance to carry out actively basic research related to VSV for the prevention and control of this disease.Secondly,VSV has a wide range of cell tropism,making it an ideal model virus for the study of virus-host interaction,viral vaccine vector and other aspects,and more progress has been made in related fields.Although VSV has been widely studied and applied as a viral vector,many researchers believe that it has potential biosecurity risks as a live viral vector.In particular,the invasion of VSV on the central nervous system(CNS)has become the biggest obstacle to the clinical application of VSV vector.In addition,in the previous work,yeast two-hybrid technique,GST-pull down and laser confocal assay were used to verify that VSV matrix protein(M)can interact with host cell translation initiation factor eIF3 i,but it is not clear how it plays the regulatory role of eIF3 i on VSV in the process of replication in the nerve cells and what effect it has on VSV replication and proliferation.First,apoptosis is closely related to the replication and proliferation of the virus.In this study,fluorescence microscopy showed that 12 or 24 h after VSV infection of nerve cells(Neuro-2a,N2a),the nuclei of the infected cells were fragmented,chromatin was dense and highly stained,and blue fluorescence was enhanced.Meanwhile,flow cytometry was used to detect the apoptosis of N2 a cells infected by VSV.The results showed that the apoptosis rate of N2 a cells infected by VSV increased significantly after 8 h compared with the control group,indicating that VSV could induce apoptosis of nerve cells.Western blotting was used to detect the activation of Akt protein kinase and its downstream key pro-apoptotic protein Bax expression in vsv-infected N2 a cells at different time points.The results showed that VSV could inhibit the phosphorylation of Akt(p-akt)and promote the expression of Bax,indicating that VSV could induce the apoptosis of N2 a cells through the Akt/Bax signaling pathway.Sceondly,in order to clarify the effect of VSV infection on eIF3 i expression,this study used qRT-PCR and Western blotting methods to respectively study the expression of eIF3 i in VSV-infected nerve cells in the gene and protein levels.The results showed that VSV(MOI=10)infecting with N2 a cells promoted eIF3 i expression at 4 h or 8 h after infection,but inhibited eIF3 i expression at 12 h.These results suggest that early VSV infection promotes the expression of eIF3 i,while late VSV infection inhibits the expression of eIF3 i.The above studies showed that the expression of eIF3 i changed during the VSV-induced apoptosis of nerve cells.To further study the effect of eIF3 i changed during the VSV-induced apoptosis of nerve cells.This study first designed and synthesized siRNA targeting eIF3 i or constructed eIF3 i overexpression vector,transfected N2 a cells were inoculated with VSV,and the virus content was detected by Western blotting,fiuorescence quantitative PCR,TCID50 and other methods.The results showed that interference of eIF3 i expression promoted the replication of VSV,while overexpression of eIF3 i inhibited the replication of VSV.To further investigate the mechanism of effect of eIF3 i on VSV replication,Western blotting was used to detect the activation of Akt after VSV infecting N2 a cells,and it was found that overexpression of eIF3 i can up-regulate p-Akt expression,Western blotting was used to detect the activation of Akt after VSV infecting N2 a cells,and it was found that overexpression of eIF3 i can up-regulate p-Akt expression,while interference with eIF3 i significantly inhibited the expression of p-Akt.Secondly,Akt specific activator SC79 was used to activate the Akt signaling pathway in N2 a cells,and then the virus was inoculated.It was found that after Akt was activated,VSV replication was significantly inhibited.The above results demonstrated that the expression of eIF3 i was closely related to the activation of Akt,and eIF3 i could promote the phosphorylation of Akt.eIF3 i can regulate apoptosis and inhibit the replication of VSV.Furthermore,qRT-PCR was used to detect the expression of Irf3,Irf7 and IFN? genes after interfering or overexpressing eIF3 i in nerve cells infected with VSV,and the expression level of interferon-related genes was significantly down-regulated after interfering eIF3 i.After eIF3 i overexpression,the expression level of interferon-related genes was significantly up-regulated.The above results showed that eIF3 i could promote Akt activation,regulating apoptosis,interfering the release of virions,and simultaneously promoting the expression of interferon-related genes to initiate an antiviral response for inhibiting the replication and proliferation of VSV.In summary,VSV-infected nerve cells regulate VSV replication and proliferation by interacting with eIF3 i and affecting its expression,regulating cell apoptosis through the Akt pathway and influencing the antiviral response.This study not only revealed the mechanism of eIF3 i in the process of VSV replication,but also may provide a theoretical basis for the prevention and treatment of anti-VSV in the central nervous system.