Preliminary Study On Interaction Between SPPV-E3L Protein And PKR In Hela Cells

The symptoms of sheep pox caused by sheep pox virus(SPPV)are similar to small pox,so itis also called"sheep variola".At present,the pathogenesis of SPPV and the immune mechanism of the organism are not clear.Virus biological characteristics and pathogenic mechanism of pox virus are the main reference model of virus vaccinia virus(VACV).Double stranded RNA protein kinase(PKR)is one of the key molecules in the host antiviral pathway,and a large number of studies show that vaccinia virus encoding E3L protein can directly or indirectly through the interaction with PKR and inhibition of PKR phosphorylation.There is a homologous gene in SPPV genome which is highly similar to VACV E3L gene sequence,but there are few studies about SPPV K3L gene,and its specific function is still unknown.Therefore,the interaction between SPPV E3L protein and host PKR was preliminarily studied in this thesis.Firstly a fluorescence quantitative detection method for the SPPV genome was established,the method has strong specificity and high sensitivity(detection limit of 10~1copies/?L)and repeatability(coefficient of variation between groups within the group of repeatability test were less than 3%)features to be used for the detection of clinical samples.Then the preservation of SPPV virus titer was measured and plotted in this study using the SPPV one step growth curve,the results showed that inoculation of 0-12h virus copy number almost no change,in a logarithmic amplification of 12-72h virus,84h into the platform.The level of activation of PKR and its downstream molecules in SPPV infected Hela cells at different time points was detected by Western-blot.The results showed that SPPV infection caused by PKR and its downstream molecules are time dependent activation.The interaction between the transient expression of E3L protein of E3L and PKR by Co-immunoprecipitation and in the cell;in order to determine the effect of verification of SPPV-E3L protein on PKR-e IF2 alpha phosphorylation and key functional sites,the establishment of SPPV-E3L,SPPV-mE3L(based on SPPV-E3L 135,168 and 174 in point mutations),and VACV-E3L ORFV-E3L eukaryotic expression vector and transient expression in Hela cells.The present study demonstrated that SPPV-E3L protein could not inhibit phosphorylation of PKR in Hela cells,and mutations in 3 critical sites,135,168 and174,could affect this function.Transient expression of VACV-E3L protein in Hela cells can increase the proliferation level of SPPV.Immunoprecipitation tests identified 4 proteins that were physically related to PKR;VACV-E3L,ORFV-E3L and SPPV-mE3L proteins inhibited phosphorylation of PKR in Hela cells,whereas SPPV-E3L protein did not inhibit PKR phosphorylation.Transient expression of VACV-E3L protein can affect the replication of SPPV.Finally,the PKR gene knockout Hela cell line was constructed by Crispr/Cas9 technique,and the Hela cell line of PKR deletion was obtained.The PKR gene translation was terminated early and PKR was nonfunctional.This study laid the foundation for further studying the biological function of SPPV and E3L and the antiviral mechanism of PKR.