Role And Mechanism Of Cholesterol Transport Homeostasis Regulated By NPC1 In Viral Replication

Lipids are important structural components of biological membranes and have important biological functions.Cholesterol is the main component of lipids,which can regulate membrane fluidity,the function of membrane proteins,and participate in membrane transport and transmembrane signal transmission.Cholesterol homeostasis plays an important role in regulating the body's metabolic balance.It has been reported that host cholesterol metabolism regulates the replication of many viruses,such as influenza virus,SARS coronavirus,Herpes simplex virus type 1 and so on.Cholesterol metabolism is involved in different stages of the virus life cycle,such as virus entry,uncoating,genome replication,assembly,and release of progeny virus particles.Exploring the relationship between viral infection and host cholesterol metabolism can not only provide an in-depth understanding of the molecular mechanism of viral replication,but also facilitate the development of host-targeted antiviral drugs and provide a theoretical basis for disease prevention and control.In this study,the fluorescent reporter viruses PRV-GFP and VSV-GFP were used to screen the existing small molecule compound library targeting host lipid metabolism in our laboratory,and we found that the small molecule compound U18666A had a significant inhibitory effect on both viruses.U18666A is an inhibitor of NPC1(Niemann-Pick type Cl).It is known that NPC1 is mainly involved in intracellular cholesterol transport.Therefore,we use U18666A to study the mechanism of cholesterol transport protein NPC1 regulating virus replication.The results are as follows:1.Inhibition of cholesterol transport regulated by NPC1 inhibits virus replicationTo explore whether U18666A exerts its antiviral effect by inhibiting NPC1 function,we used NPC1 knock-down and knock-out cell lines,and found that NPC1 deletion significantly inhibits PRV and VSV replication.Adding U18666A to NPCl knock-out cells cannot further inhibit virus replication shows that U18666A exerts antiviral effect by inhibiting NPC1.Further research found that U18666A can significantly inhibit the proliferation of DNA viruses(PRV and HSV)and RNA viruses(PRRSV,VSV,AIV,and MHV),indicating that cholesterol transport regulated by NPC1 plays an important role in viral replication.2.The cholesterol transport disorder regulated by NPC1 interferes with the kinetics of CCPs by reducing plasma membrane cholesterol,thereby inhibiting virus replicationThe viral infection stage is divided into adsorption,entry,transcription,replication and release.These experiments such as genome labeling,cell surface biotinylation assay,qRT-PCR,electron microscopy confirmed that inhibiting cholesterol transport regulated by NPC1 can inhibit the entry stage of the virus.It is known that there are two main ways for viruses to enter cells:membrane fusion and endocytosis.Cell endocytosis is mainly divided into CME(Clathrin-mediated endocytosis),caveolin-mediated endocytosis,macropinocytosis and phagocytosis,and other unknown forms of endocytosis.We found inhibiting cholesterol transport regulated by NPC1 does not affect the membrane fusion between the virus and the cell membrane,and mainly inhibits the replication of the virus by affecting CME.Further experiments show that inhibiting cholesterol transport regulated by NPC1 mainly interferes with the dynamic of CCPs(Clathrin-coated Pits),and has no effect on the expression level of key components of CME,subcellular localization,and the interaction between viruses and CCPs.It is known that cholesterol determines cell membrane fluidity and phase transition,and cholesterol transport disorder regulated by NPC1 can cause cholesterol accumulation in cells.Therefore,the filipin staining experiment found that U18666A can reduce plasma membrane cholesterol.Adding exogenous cholesterol can restore CCPs' dynamics,virus entry and infection.In summary,it is shown that inhibiting cholesterol transport regulated by NPC1 can hinder intracellular cholesterol transport and reduce plasma membrane cholesterol,leading to defects in CCPs dynamics,thereby inhibiting virus entry and exerting a broad-spectrum antiviral effect.3.Cholesterol transport protein inhibitor U18666A has a protective effect on PRV and H1N1 infected miceTo detect the antiviral effect of inhibiting cholesterol transport regulated by NPC1 in mice,mice were infected with PRV and H 1N1 after intraperitoneal injection of U18666A.The results showed that U18666A can inhibit the replication of PRV in the brain and reduce the damage of H1N1 to the lung.U18666A treatment can significantly reduce the inflammatory response caused by viral infection and improve the survival rate of mice.This shows that the use of cholesterol transport inhibitors has a certain protective effect on virus-infected mice.In summary,we found that inhibiting cholesterol transport regulated by NPC1 can inhibit viral replication,indicating that targeting plasma membrane cholesterol levels or interfering with host CCPs' dynamics to inhibit viral entry is an effective target for the development of antiviral drugs.This study provides new ideas for strengthening the in-depth understanding of viral replication mechanisms and development of broad-spectrum antiviral drugs.