Preliminary Study On The Mechanism Of HnRNP K Gene Inhibiting BEFV Replication

Bovine ephemeral fever?BEF?is an acute,febrile bovine infectious disease caused by bovine ephemeral fever virus?BEFV?.It can lead to the decrease of milk production and the increase of mortality,and cause significant economic losses to the cattle industry in China.Although many commercial vaccines have been produced to prevent BEFV infection,unfortunately,current preventive and control strategies cannot provide sustainable protection for cattle infected with BEFV.In recent years,the interaction mechanisms between host factors and viruses have attracted more and more attention.Therefore,in-depth study of the molecular mechanism of BEFV replication and research on the interaction between viruses and host factors will help us to better understand the mechanism of viral infection.The BEFV genome encodes five structural proteins?N,P,M,G,and L?,and six non-structural proteins(G_NS,?1,?2,?3,?,?).Studies have shown that viral proteins play an important role in viral replication,but the function of non-structural proteins of BEFV is largely unknown.Therefore,by studying the function of the non-structural protein of BEFV,it will lay the foundation for the in-depth discussion of the molecular mechanism of BEFV replication.hnRNP K is a member of the hnRNPs family.It can interact with RNA/DNA and multiple proteins,and participate in the biological process of RNA shuttling,transcription,translation regulation,cell proliferation and apoptosis.Current studies have shown that hnRNP K affects the replication of many viruses.However,the interaction between BEFV and hnRNP K has not been reported.In this study,the interaction of host gene hnRNP K,viral protein?3 and apoptosis was studied,and the main research progress was as follows:Through gene overexpression and silencing technology,it was found that hnRNP K gene inhibited the replication of BEFV.Firstly,the shRNA silencing expression vector of hnRNP K and hnRNP K silencing cell lines were constructed respectively.After infection with BEFV,it was found that knockdown of hnRNP K gene promoted BEFV replication by detecting the virus titer.At the same time,hnRNP K lentivirus overexpression vector and hnRNP K overexpression cell lines were constructed.After BEFV infection,it was found that overexpression hnRNP K could inhibit the replication of BEFV.The results of co-immunoprecipitation showed that hnRNP K interacted with viral protein?3.Overexpression of hnRNP K inhibited?3 expression independent of proteasome,autophagy lysosome and caspase pathway.The interaction between hnRNP K and?3 was confirmed by protein immunoprecipitation.BHK-21 cells were co-transfected with?3 eukaryotic expression vector and hnRNP K eukaryotic expression vector and control vector respectively.It was detected that overexpression of hnRNP K suppressed the expression of viral protein?3.After treatment with MG132,CQ,Z-VAD-FMK,Z-IETD-FMK,the expression level of?3 did not callback,indicating that hnRNP K inhibits the expression of viral protein?3independent of proteasome,autophagy lysosome and caspase pathway.The apoptosis was induced by the non-structural protein?3 of BEFV,which promoted the replication of BEFV,and the apoptosis induced by BEFV was inhibited by hnRNP K.Overexpression of?3 gene increased the expression of Cleaved caspase3 and Cleaved PARP,suggesting that BEFV non-structural protein?3 can induce apoptosis.Subsequently,BHK-21 cells were pre-treated with apoptosis inhibitor Z-VAD-FMK and apoptosis inducer CCCP respectively,DMSO group as control.The BEFV of 0.1MOI infected the treatment group and the control group for 24 h,and the virus titer was detected by TCID₅₀.The results showed that apoptosis promoted the BEFV replication.BEFV infected hnRNP K overexpression cell lines,the expression of Cleaved caspase 3 and Cleaved PARP was significantly downregulated compared with the control group,indicating that overexpression of hnRNP K inhibited BEFV-induced apoptosis.The expression of hnRNP K protein was down-regulated by BEFV infection.The degradation of hnRNP K was not via proteasome and autophagy lysosome pathway,but mainly mediated by caspase 3 activity.RT-qPCR and western blot were used to detect the samples of BHK-21 cells infected by BEFV at different time points.It was found that hnRNP K mRNA level was up-regulated and protein level was down-regulated after BEFV infection.After MG132 and CQ treatment,the expression level of hnRNP K protein did not increase,indicating that BEFV downregulated the expression of hnRNP K protein not through ubiquitin proteasome and autophagy lysosome pathway.Then,BHK-21 cells were pretreated with caspase 3 inhibitor Z-DEVD-FMK for 4 h and infected with BEFV,and found that the expression of hnRNP K protein was reversed.The results showed that the degradation of hnRNP K was mediated by caspase 3 activity.In conclusion,this study explored the interaction between host gene hnRNP K,viral protein?3 and apoptosis.It was discovered that BEFV infection down-regulated the expression of hnRNP K protein,hnRNP K inhibited viral replication,and viral protein?3 induced apoptosis and apoptosis promoted the replication of BEFV.It was first revealed that hnRNP K suppressed BEFV replication by inhibiting the expression of?3 and the apoptosis induced by BEFV.The down-regulation of hnRNP K protein was mediated by activation of caspase 3.It provides a theoretical basis for the development of antiviral drugs.