The Effects Of HSP27?70?90 On PRV Infection In PK-15 And The Mechanism

Porcine pseudorabies?PR?is an acute and febrile infectious disease caused by porcine pseudorabies virus?PRV?in many mammals and some birds.PR is an outbreak epidemic and can co-infect with many other pathogens and cause complications,which threatens the pig industry all over the world.At present,several researches have shown that heat shock proteins?HSPs?are differentially expressed in host-cells infected with various viruses,and revealed that HSPs participate in the synthesis of viral proteins,viral particles,protein folding and aggregation,and also play an important role in antigen presentation.In our previous studies,we also detected that several HSPs were differentially expressed in PK-15 cells infected with PRV using roteomic analysis,but the roles of these HSPs in PRV infection are unclear.In the present study,the effects of PRV infection on the proliferation of host cells and the expression of HSP27,HSP70 and HSP90 were studied;PK-15 cell strains with stable down-regulation of HSP27,HSP70 and HSP90 were constructed by lentivirus interference vector;then the effects of down-regulation of HSPs on PRV infection including virus virulence,replication,expression and localization,cell proliferation and apoptosis,and the expression of some antiviral factors were explored in the three HSP down-regulated cells,respectively.The results obtained are as following:1.PRV infection inhibited proliferation of PK-15 cells in a time and concentration-dependent manner by monitored iCELLigence Real-Time Labeled Cell Function Analyzer.Low viral titers infection?no more than 10 TCID50?showed no significant effect on cell proliferation within 72 h after PRV infection,while high viral titers(more than 20 TCID₅₀)significantly inhibited the proliferation of PK-15 cells.The viral nucleic acid content was significantly increased after PRV infection detected by Real-Time qPCR,and the expression of HSP27,HSP70 and HSP90 was significantly increased at early stage of PRV infection with 10 TCID50 using Real-Time qPCR and Western-blot assays.2.The lentiviral vector of shRNA-HSP27,shRNA-HSP70 and shRNA-HSP90was constructed and transfected into packaging cells to obtain the corresponding lentivirus.To establish the stable down-regulated expression of HSP27,HSP70 and HSP90 of cell lines,PK-15 cells were infected with the lentivirus targeting the HSP27,HSP70 and and HSP90 followed by screening with puromycin.The expressions of the three HSPs were verified by RT-qPCR and WB.3.In the stable down-regulated expression of HSP27,HSP70 and HSP90 of PK-15 cell lines:?1?The viral titers were significantly decreased by Reed-Muench method when compared to that in the control cells,as well the expression of the PRV gE gene;?2?PRV antigen was mainly located in the cell nucleus,and the fluorescence signal of PRV in three HSP-interfering cells was significantly weakened through detection of immunocytochemistry?ICC?;?3?The proliferation of HSPs down-regulated cells was inhibited to some extent after PRV infection;?4?The level of Bax in three shRNA-HSPs cell lines was increased significantly at different time points after PRV infection,and the expression of Bcl-2 was decreased;?5?The expressions of the anti-virus factors including IFN?,Mx1 and RNaseL were promoted to some extent after down-regulation the expression of three HSPs in PK-15 cells.The results indicate that PRV infection can inhibit the proliferation of PK-15cells,and induce the rapid stress response in PK-15 cells with increasing the expression of HSP27,70 and 90.Down-regulation of HSP27,HSP70 and HSP90 can inhibit the PRV infection in PK-15 cells,which may be achieved via reducing the viral titer,inhibiting the cell proliferation and promoting cell apoptosis and the expression of the related antiviral factors.These data can provide a theoretical basis for the development of HSPs as potential anti-PRV drugs.