Molecular Mechanism Of Host Protein CH25H Regulation The Replication Of Encephalomyocarditis Virus

Encephalomyocarditis virus(EMCV)is an important zoonotic pathogen with a wide range of hosts,which can cause encephalitis,myocarditis,diabetes,reproductive system diseases and other diseases.EMCV can spread across species,seriously endangering the health of pigs and public health safety.Cholesterol 25-hydroxylase(CH25H)is a polyectopic membrane protein encoded by an important interferon-stimulating gene(ISG)that can oxidize cholesterol to 25-hydroxycholesterol(25HC).Early studies have shown that CH25H and 25HC can significantly inhibit the replication of many viruses inclouding a variety of enveloped and non-enveloped viruses,but the effect of CH25H on EMCV replication and the mechanism are currently unclear.In this study,the real-time PCR(qPCR)method was used to detect the change of CH25H mRNA level at different time points after EMCV infection on BHK-21 and N2A cells.The CH25H eukaryotic expression plasmid was constructed,and its effect on EMCV replication was detected by transfection in vitro.It has laid a foundation to study the infection and replication mechanism of EMCV and the antiviral mechanism of CH25H.The specific research content is as follows:1.The Molecular Mechanism of CH25H Inhibits EMCV Replication in BHK-21 cellsBHK-21 cells were used to be infected with EMCV before cell lysates collection at different hours post-infection(h.p.i).The mRNA levels of CH25H were analyzed by qRT-PCR.The results showed that EMCV can significantly promote the transcription of CH25H mRNA after infection,indicating that EMCV can induce CH25H gene expression in BHK-21 cells.In order to study the effect of CH25H on EMCV replication in BHK-21 cells,the expression of CH25H was regulated by transfection of eukaryotic expression plasmids and siRNA interference fragments.The cell samples were analyzed by Western blot,indirect immunofluorescence(IFA)and qPCR methods.The results showed that CH25H can significantly inhibite EMCV replication in a dose-dependent manner.By constructing a mutant CH25H-M with missing enzyme activity and transfecting it into BHK-21,it was found that the mutant's activity in inhibiting EMCV was significantly reduced.In addition,this study found that the enzyme active product 25HC also has significant anti-EMCV activity.We then studied the effect of 25HC on the replication cycle of EMCV.Through analysis of various aspects of virus particle adsorption,penetration,replication and release,we found that 25HC can significantly inhibit EMCV penetration and genome replication.BHK-21 cells were co-transfected with plasmid expression viral proteins and CH25H,it was found that CH25H can degrade RNA-dependent RNA polymerase(RdRp,3D protein),followed by laser confocal and immunoprecipitation test(co-IP)to discover 3D of CH25H and 3D protein are localized in the cytoplasm and interact specifically.The above results indicate that CH25H can inhibit the incorporation of EMCV and genome replication by producing 25HC.At the same time,CH25H can also degrade 3D protein and affect RNA polymerase to inhibit viral replication.2.The Molecular Mechanism of CH25H Inhibits EMCV Replication in N2A cellsThe N2A cell line is a mouse brain neuroma cell.In order to study the effect of CH25H on EMCV infection in N2A cells,qPCR was used to detect the mRNA level of CH25H in the cells after EMCV infection.The effect of upregulation was most obvious after 16h,indicating that EMCV can induce CH25H gene expression in N2A cells.Western blot,qPCR and virus titer determination experiments(TCID50)were used to analyze the effect of overexpressing CH25H on EMCV replication.The results showed that CH25H could significantly inhibit EMCV replication in N2A cells in a dose-dependent manner.Through transfection of the mutant CH25H-M,it was found that the mutant's inhibitory effect on EMCV was significantly reduced.In addition,the N2A cells were pretreated with 25HC and found that 25HC can also significantly reduce the virus titer of EMCV.Through analysis of various aspects of virus particle adsorption,penetration,replication and release,we found that 25HC can significantly inhibit EMCV entry into N2A cells and it's genome replication.The above results indicate that CH25H and 25HC can significantly inhibit EMCV infection in N2A cells.Further studies have found that 25HC significantly inhibits EMCV replication on N2A cells by inhibiting viral particle entry and genome replication.