Molecular Mechanism Of Cholesterol And Fatty Acid Homeostasis On Classical Swine Fever Virus Infection Of PK-15 Cells

Classical swine fever(CSF)is a highly contagious infectious disease caused by classical swine fever virus(CSFV),which has caused severe economic losses to the global pig industry.The only natural hosts are domestic pigs and wild boars.Different strains of CSFV have different virulence,but all of them will cause great harm to pig industry,so it is urgent to find new and effective anti-virus strategies.Studies have shown that homeostasis of cholesterol and fatty acids is essential for viral replication,and viruses can recombine lipid metabolism of host cells,thus creating a favorable environment for virus reproduction,so the study of cholesterol and fatty acids can provide new ideas for antiviral therapy.In this experiment,the cholesterol transport inhibitor U18666A was used to interfere with intracellular cholesterol transport and cause cholesterol accumulation in lysosomes,thereby effectively inhibiting the swine fever virus membrane fusion process.In addition,through confocal and coimmunoprecipitation experiments,it was proved that fatty acid synthase interacts with the non-structural protein CSFV-NS4B of classical swine fever virus,and regulates the replication of classical swine fever virus.The research content is as follows:1 U18666A Inhibits Classical Swine Fever Virus Infection by Interfering Membrane FusionFirstly,we used CSFV to infect PK-15 cells,and then used different concentrations of U18666A to maintain them,collected the samples at 12h and 24h respectively.The expression level of virus proteins can be identified by Western Blot and Confocal,and the copy number of CSFV was quantified by RT-qPCR.The results showed that when U18666A disrupted the transportation of cholesterol,the CSFV replication would be affected in a time-and concentration-dependent manner.Secondly,PK-15 cells were treated with U18666A at different time points of CSFV infection,the RT-qPCR results showed that U18666A affects the fusion and replication stages of the virus life cycle,but does not affect its binding and entry.Finally,the effects of U18666A on CSFV,cholesterol and lysosomes were observed by Confocal.The results showed that U18666A caused accumulation of intracellular cholesterol in lysosomes,loss of lysosome function,and CSFV could not carry out membrane fusion.In conclusion,this experiment proves that U18666A can effectively inhibit CSFV replication and is a potential drug candidate for anti-pestivirus treatment.2 NPC1 Protein Regulates Classical Swine Fever Virus ReplicationIn order to study the role of NPC1(a lysosome transport protein)in the replication of classical swine fever virus,we first transfected siRNA targeting NPC1 into PK-15 cells and inoculated CSFV,the knockdown efficiency was confirmed by Western blot and RT-qPCR,and the effects on cholesterol transport,lysosome content,and CSFV replication after knockdown were observed.In addition,through the same treatment as U18666A,we verified the antiviral effect of imipramine.Finally,Vorinostat was added to U18666A-treated PK-15 cells and infected with CSFV.The effect of Vorinostat on CSFV protein expression and cholesterol was observed by Confocal.Our results show after PK-15 cells were treated with U18666A,intracellular cholesterol accumulated in lysosomes and inhibited CSFV replication.Imipramine(a cationic hydrophobic amine similar to U18666A)also inhibits CSFV replication through a similar mechanism.Unexpectedly,histone deacetylase inhibitor(HDACi)Vorinostat reversed the antiviral effect of U18666A,indicating that HDACi reversed the dysfunction of NPC1,the accumulation of intracellular cholesterol disappeared,and CSFV replication was restored.In conclusion,our studies have shown that NPC1 protein is essential for CSFV replication.3 FASN interacts with CSFV-NS4B to promote CSFV proliferationTo study the role of fatty acid synthase in PK-15 cells infected with CSFV,we used small molecule inhibitors to inhibit fatty acid synthesis and observed virus replication.First,C75,TOFA or a combination of two drugs were used to treat PK-15 cells,in addition,palmitic acid and malonyl-coenzyme A was added to C75/TOFA-treated cells,and then infected with CSFV,the copy number of virus was quantified by RT-qPCR.Second,C75 was added at different time points of CSFV infection,the effects of C75 on CSFV life circle were determined by RT-qPCR.Later,we transfected non-structural proteins of CSFV into PK-15 cells,and the interactions between virus proteins and FASN were detected by Confocal and co-immunoprecipitation.Finally,PK-15 cells were infected by CSFV,and the positional relationship of dsRNA,FASN and each organelle was observed by Confocal.Our results show that C75 and TOFA can effectively inhibit CSFV replication by interfering with fatty acid metabolism.The addition of malonyl-CoA and palmitic acid,completely restored the inhibitory effect of FASN inhibitors.C75 affects the replication and release phases of the CSFV life cycle,but does not affect its binding and entry.The results of Confocal and COIP showed that fatty acid synthase can interact with CSFV-NS4B and promote virus biosynthesis on the endoplasmic reticulum.In short,fatty acid synthase promotes CSFV replication,and the results provide reference for finding new antiviral targets.