The Effect Of CH25H Gene On BPIV3 Replication And The Study Of Its Mechanism

Bovine parainfluenza is an acute and contact-induced respiratory infectious disease caused by bovine parainfluenza virus type 3?BPIV3?,which mainly infects the respiratory organs of cattle and can cause the decline of the body's immune function.The virus spread rapidly and had a high incidence.Most of the diseased cattle manifested as clinical symptoms of loss appetite,runny nose,cough,fever and dyspnea.BPIV3 could be mixed with other viruses,resulting in Bovine respiratory disease complex?BRDC?,which has greatly increased the mortality of diseased animals,and seriously threatened the development of cattle industry around the world.At present,developed countries in the dairy industry mainly use inactivated and attenuated vaccines to prevent the disease.However,the development of related vaccines and diagnostic reagents in China has lagged behind,and there are no specific prevention and treatment measures for the disease.Therefore,the main epidemic strains in China will be taken as the research objects to in-depth study of the pathogenic mechanism of BPIV3,screening drug targets,will provide a theoretical basis for accelerating the development of effective antiviral drugs and parainfluenza vaccines.Cholesterol-25-hydroxylase?CH25H?is a 31.6 kDa multi-transmembrane and endoplasmic reticulum?ER?-associated enzyme whose uses O₂ as an additional substrate and NADPH as a cofactor that catalyzes the oxidation of cholesterol to produce 25-hydroxycholesterol?25HC?to reduce the accumulation of cholesterol.Recent studies have found that CH25H is an interferon-stimulated gene?ISG?,which taken part in the antiviral innate immunity.CH25H mainly exerts its antiviral effect through its enzyme activity product 25HC,and can also inhibit the replication of some viruses in a manner independent of its enzyme activity.However,the antiviral effects and mechanisms of CH25H and 25HC for paramyxovirus including BPIV3 remain unclear.To explore the effects of BPIV3 infection on the expression of CH25H and the effect of bovine CH25H on BPIV3 replication,we carried out the exploring as the following:?1?MDBK cells and Hela cells were infected with BPIV3.It was found that the expression of CH25H mRNA was up-regulated by BPIV3 infection,whereas CH25H protein expression was down-regulated in the later stages of BPIV3 infected MDBK cells and Hela cells by qRT-PCR and Western Blot assays.?2?Quantitative detection primers for IFN-?,CH25H and ISGs were designed respectively,and the MDBK and Hela cells were treated with poly?I:C?or IFN-?,the effects of these molecules on CH25H mRNA expression were verified by qRT-PCR assays.The results showed that CH25H is an ISG in MDBK cells and Hela cells.In order to verify the down-regulated mechanism of CH25H protein expression,the proteasome inhibitor MG132,the autophagy inhibitor CQ,the apoptosis inhibitor Z-VAD-FMK and the protein synthesis inhibitor CHX were added to Hela cells infected with BPIV3 and viral genes were overexpressed in Hela cells.The results indicated that BPIV3 destroyed the stability of CH25H protein and thereby inhibited the expression of CH25H protein,it was not related to the major intracellular protein degradation pathways in eukaryotic cells.?3?The eukaryotic expression vectors of CH25H gene were constructed.Overexpression and silencing of CH25H gene in MDBK and Hela cells,respectively.The changes of virus titer and viral protein HN expression were detected after BPIV3infection.The results suggested that overexpression of CH25H significantly inhibited BPIV3 replication,while silencing CH25H has the opposite effect.?4?The MDBK cells were pretreated with the enzyme activity product 25HC of CH25H.After BPIV3 infection,the cell samples were collected at the corresponding time.The copy number of the virus genome N was detected by qRT-PCR absolute or relative quantification.It was found that 25HC could inhibit the synthesis of BPIV3cRNA and gRNA in MDBK cells,and the CH25H mutant?CH25H-M?lacking enzyme activity also has some antiviral effects after mutation of the enzyme activity site of CH25H.The above results indicate that CH25H inhibits BPIV3 replication through enzyme activity-dependent and-independent ways.In here,MDBK cells were used as the main pattern cells.We have preliminarily demonstrated the antiviral effect of host gene CH25H on BPIV3,and revealed its molecular mechanism,which provided clues and target molecules for the development of anti-BPIV3 drugs.