The Molecular Mechanism Of Cholesterol-25-hydfroxylase Inhibiting The Replication Of Seneca Valley Virus

Seneca Valley virus(SVV)is a single stranded RNA virus without envelope,belonging to the Picornaviridae family Seneca virus.In recent years,it has been found that SVV infection is associated with sporadic vesicular disease(PIVD)in pigs.Pigs of all ages are susceptible.The main clinical manifestations are lameness and blisters.The mortality rate of newborn piglets within 7 days is as high as 30%-70%.As a new infectious disease,there are no effective commercial vaccines and antiviral drugs.Therefore,it is urgent to carry out antiviral research on SVV and seek effective prevention and treatment methods.Cholesterol-25-hydroxylase(CH25H)is an interferon stimulated factor(ISG),which catalyzes the addition of a second hydroxyl group to the 25 th carbon atom of cholesterol to produce 25-hydroxycholesterol(25HC).Recent studies have shown that CH25 H and 25 HC are broad-spectrum antiviral host factors and play an important role in host antiviral innate immune response.In order to investigate the effect of CH25 H on SVV proliferation and its related molecular mechanism,the following research contents were carried out with SVV-HB as the research object.1.The effect of CH25 H on SVV-HB replicationCH25H is an interferon stimulated factor.It is not clear whether CH25 H plays a regulatory role in the replication of interferon sensitive SVV-HB.To evaluate the effect of SVV-HB infection on the expression of endogenous CH25 H,HEK-293 T and BHK-21 cells were infected with 1.0 MOI SVV-HB,respectively.The cells were collected at different time points,and the protein and m RNA expression levels were detected by western blot and q RT-PCR.The results showed that the protein expression level of CH25 H decreased continuously at different time points after SVV-HB infection,and the percentage of protein decrease reached 48% at 12h;the m RNA expression level of CH25 H decreased significantly(* * P < 0.001).Therefore,SVV-HB may play a role in the pathogenesis of host infection by down regulating the expression of CH25 H.In order to determine the effect of CH25 H on the proliferation of SVV-HB,HEK-293 T cell line overexpressing mCH25H(mouse CH25H)was infected with 1.0MOI SVV-HB.The proliferation of SVV-HB was analyzed by western blot,q RT-PCR and viral plaque assay.The results showed that compared with the control group,the expression level of VP1 protein in the mCH25 H expression group was decreased;the relative decrease level of VP1 m RNA was extremely significant at 9h after infection(*P<0.001),and the relative viral titer was reduced about three times(*P<0.001).Further study showed that mCH25H inhibited SVV-HB proliferation in a dose-dependent manner.In view of the potential of SVV-HB in human oncolytic therapy and swine epidemic therapy,the effects of human(h CH25H)and pig(s CH25H)on the proliferation of SVV-HB were evaluated in HEK-293T cells.The results showed that both of them could inhibit the proliferation of SVV-HB,indicating that there was no species-specific inhibitory effect of CH25H on SVV-HB.In order to further determine the inhibitory effect of CH25 H on the proliferation of SVV-HB,sh RNA was used to knock down the expression of CH25 H in HEK-293 T cells and then infect SVV-HB.The results showed that the expression of VP1 protein was up-regulated compared with the control group,indicating that silencing the expression of CH25 H gene can promote the proliferation of SVV-HB.2.Molecular mechanism of CH25 H inhibiting SVV-HB replicationThe above results showed that CH25 H can effectively inhibit the proliferation of SVV-HB,and CH25 H can affect the proliferation of different viruses through different mechanisms.In order to explore the molecular mechanism of CH25 H inhibiting the replication of SVV-HB,we first evaluated the role of CH25 H enzyme activity in its inhibition of SVV-HB proliferation.A mutant(mCH25H-M)lacking hydroxylase activity of mCH25 H was constructed.mCH25 H,mCH25H-M and control plasmids were transfected into HEK-293 T and BHK-21 cells respectively.The cells were infected with SVV-HB for 9 hours,and the samples were collected by Western blot.The effect of mCH25H-M on SVV-HB proliferation was evaluated by blot and viral plaque assay.The results showed that mCH25H-M had no effect on SVV-HB proliferation without hydroxylase activity,indicating that CH25 H inhibited SVV-HB proliferation depending on its enzyme activity.Studies have shown that CH25 H can affect the proliferation of SVV-HB by catalyzing cholesterol to produce 25 HC.In order to explore whether CH25 H can affect the proliferation of SVV-HB by relying on 25 HC,BHK-21 cells were directly treated with 25 HC,and then infected with 1 MOI SVV-HB after selecting the appropriate concentration of 25 HC.These results of blot showed that the expression level of VP1 protein decreased 2-5 times after 25 HC application.The inhibitory effect of 5?mol /L 25 HC on SVV-HB was the strongest,and the expression level of VP1 m RNA and viral titer were significantly decreased(* * P < 0.001).In order to further explore the molecular details of 25 HC inhibiting SVV-HB,we further analyzed whether 25 HC can directly kill virus particles.Through virus inactivation experiment analysis,we found that 25 HC can not directly kill virus particles.Furthermore,the role of 25 HC in virus replication was analyzed.Through the virus adsorption test,entry test,replication test and release test,it was found that25 HC only played an effect in the virus adsorption stage.Compared with the control group,the virus titer and m RNA level of 25 HC group were significantly reduced(* *P < 0.001).This study showed that CH25 H of different species could effectively inhibit the proliferation of SVV-HB,and revealed the molecular mechanism of CH25 H inhibiting the proliferation of SVV-HB.These results provide new ideas for the follow-up SVV infection immunity and antiviral drug design,and lay the foundation for the development of new vaccines and oncolytic virus design.