The Mechanism Of BmNPV Protein And Cholesterol Media BmNPV Infection

Cholesterol plays an important role in various biological processes such as cell differentiation,embryonic development,immune response,and neuronal signaling.In the late endosome low pH environment,cholesterol is released from lipoprotein and transported to the cytoplasm through NPC1 protein-mediated transmembrane transport after being transferred by NPC2 protein.Mutations in NPC proteins can lead to Niemann-Pick disease in humans,characterized by cholesterol accumulation in cells.NPC proteins and cholesterol not only play important roles in cellular physiology but are also exploited by many viruses to infect cells.Many highly pathogenic enveloped viruses,including Ebola virus and hepatitis A virus,use NPC 1 protein as a receptor to release genetic material into the cytoplasm through its mediated membrane fusion process.The infection of African swine fever virus depends not only on NPC1 protein but also on the help of NPC2 protein.Cholesterol is also involved in various stages of the virus infection cycle.It may be precisely because of the importance of NPC protein and cholesterol in cell physiological activities,and the relatively conservative cholesterol transport pathway in the organism,that it has been hijacked by a variety of viruses.In the mid-19th century,baculovirus was identified in infected silkworms,unlike other viruses,there are two virion forms:one is an embedded virion(OCLUSIONderived virus,ODV),in the oral infection of the virus,the polyhedral shell of ODV is dissolved in the alkaline environment of the arthropod intestine,becoming a virus with infectious ability and infecting intestinal cells;The other is budded virus(BV),which causes cell-to-cell systemic infection and enters host cells through clathrin-mediated endocytosis.In addition,as an important biological tool,baculovirus play an important role in many fields such as drug development,vaccine production,and recombinant virus insecticides.Baculovirus have been discovered for more than a hundred years,but the receptor for virus infection of cells and the specific molecular mechanism have not been elucidated.Previous studies have found that the BmNPC1 protein plays an important role in the process of BmNPV(Bombyx mori nucleopolyhedrovirus)entering cells and interacts with the BmNPV envelope glycoprotein GP64.The BmPP(Bombyx mori promoting protein),a member of the NPC2 protein family in silkworms,promotes BmNPV infection of silkworm cells and has the ability to bind to cholesterol.In addition,it has been reported that molecules such as phospholipids in cells participate in the process of baculovirus adhesion to cells.However,the mechanism by which BmNPC proteins and cholesterol mediate BmNPV infection is still unknown.Based on this,this study first explored the localization of BmNPC1 protein and its role in the process of baculovirus entering host cells,and added virus to observe the infection situation after expressing BmNPC1 in the BmNPV non-nanometer cell line Sf9 to verify whether it is the intracellular receptor for BmNPV infection of host cells.The period in which BmNPC2 protein affects viral infection was further investigated by knocking out the BmNPC2 cell line,and further functional validation was performed by adding purified BmNPC2 protein.The interaction between BmNPC2 protein,BmNPC1-C and GP64 protein was clarified by yeast double hybridization and co-immunoprecipitation experiments,and the molecular mechanism of how BmNPC protein co-mediates BmNPV infection was analyzed.Finally,the concentration of cellular cholesterol was reduced by Lov and MβCD drug treatment,and the role of cholesterol in baculovirus infection was analyzed.The main findings are as follows:1.Molecular mechanism of BmNPC1-mediated entry of baculovirus into cellsIn this study,the subcellular localization of BmNPC1 protein in Bombyx mori BmE cells was investigated using cell membrane and late endosome markers.The results showed that the subcellular localization of BmNPC1 protein is different from that of human NPC1 protein,as BmNPC1 protein is located on the cell membrane and late endosome membrane.Using BmNPC1-knockout cells,the early infection of the virus was investigated,and the results showed that the absence of BmNPC1 did not affect the adhesion of the virus to BmE cells.Two hours after viral infection,BmNPV was found to release a large amount of nucleocapsids into the cytoplasm through membrane fusion and enter the nucleus through the nuclear pore.However,in BmNPC1-knockout cells,the entry of BmNPV into the nucleus was significantly inhibited,and the nuclear localization rate was only 1/8 of that of the control BmE cells.These results suggest that BmNPC1 protein may play an important role in the membrane fusion process of BmNPV budded virus particles.Further experiments using DiO dye-wrapped recombinant BmNPV added to BmNPC1-knockout cells showed that the virus membrane fusion process was significantly inhibited.After purifying BmNPC1-C protein,it was wrapped around the virus and added to BmE cells.The results showed that the virus infection rate was only 20%of that of the control group,indicating that the interaction between BmNPC1 protein and the virus through its extracellular domain C mediates the fusion of the virus envelope.To investigate whether BmNPC1 is an intracellular receptor for BmNPV infection,BmNPC1 was expressed in non-nucleate cells Sf9.After adding BmNPV,the virus was able to release nucleocapsids through the membrane fusion barrier in Sf9 cells and enter the nucleus through the nuclear pore,but BmNPV was unable to replicate in Sf9 cells.2.Molecular Mechanism of BmNPC2 Protein Promoting Infection of BaculovirusUsing the CRISPR/Cas9 system to knock out BmNPC1 in BmE cells,staining with Filipin dye revealed an accumulation of cholesterol in cells after BmNPC2 knockout,confirming the function of BmNPC2 protein in transporting cholesterol.When recombinant BmNPV-VP39-EGFP was added to the BmNPC2 knockout cell line,it was found that BmNPC2 knockout did not affect virus binding,but it did affect the efficiency of virus entry into the nucleus.Further studies showed that BmNPC1 knockout affected the efficiency of virus membrane fusion and thus affected virus entryinto nuclear.By expressing and purifying BmNPC2 protein using the baculovirus expression system,it was found that adding BmNPC2 protein to BmE cells could promote virus infection and significantly enhance BmNPV membrane fusion efficiency,further validating previous results.When BmNPC2 was added to BmNPC1 knockout cells,it could not promote virus infection,indicating that BmNPC2’s promotion of BmNPV infection depends on BmNPC1 protein.In immunoprecipitation experiments with co-expression of BmNPC2 and BmNPC1-C protein as well as BmNPC2 and GP64 protein in cells,it was found that BmNPC2 protein could interact with BmNPC1-C and GP64 proteins,which was also shown in yeast two-hybrid experiments.When BmNPC2 protein was bound to budding virus obtained from BmNPC2 knockout cells,it could enhance the infectivity of budding virus.ELISA experiments showed that adding BmNPC2 protein could not enhance the interaction intensity between BmNPC1-C and GP64 proteins,but low pH treatment could enhance the interaction intensity between BmNPC1-C and BmNPC2 as well as between BmNPC1-C and GP64 proteins.This indicates that BmNPC2 protein may promote membrane fusion by enhancing the efficiency of interaction between BmNPC1-C and GP64 proteins.3.Mechanism of cholesterol in baculovirus infectionTo explore the role of cholesterol in BmNPV infection by adding Lovastatin,a drug that competitively inhibits the rate-limiting enzyme oxyglutaryl-CoA reductase enzyme during cholesterol synthesis,and MβCD,a drug that depletes cell membrane cholesterol,or adding exogenous cholesterol to reduce or increase intracellular cholesterol concentrations.We add different concentrations of Lov to BmE cells to detect the effect of Lov treatment on cell viability using CCK-8 reagent,and Lov adding more than 1 umol/ml to BmE cells affects cell viability.Three days after the addition of Lov inside BmE cells,the intracellular cholesterol concentration dropped to 60%of the control group.After BmE cells undergoing Lov treatment,they had a significant inhibitory effect on viral infection,and the viral DNA load in the drug-treated group was only 1/4 of that of the control group after three days.After removed membrane cholesterol using Lov and MβCD,the amount of viral binding was significantly reduced,only 50%of that of the control group.Furthermore,it was found that reducing intracellular cholesterol concentration affects the efficiency of viral membrane fusion,which in turn leads to a decrease in the amount of virus nucleation,and when the exogenous cholesterol is replenished,the virus binding and the amount of virus into nuclear are restored.After Lov treated BmE cells,the effect on viral protein expression was detected 20 hours after viral infection,and it was found that after the concentration of cholesterol decreased in the cells,it had no effect on the expression of viral proteins.Furthermore,we explored the effect of reducing intracellular cholesterol concentration on viral budding,purified the budding virions in normal cells and Lov-treated cells for Western blotting analysis,and found that the viral budding amount after Lov treatment was significantly reduced compared with the control group.The colocalization of GP64 protein and cellular lipid raft during virus budding was observed by IFA experiment,and the results showed that Lov treatment inhibited the formation of cell membrane lipid raft,which in turn led to a decrease in virus budding.In summary,BmNPC1 may be a fusion receptor for BmNPV-infected cells,which mediates the membrane fusion process of the virus,and can break the membrane fusion barrier in BmNPV-accepted Sf9 cells.BmNPC2 protein-facilitated BmNPV infection is through the intracellular cholesterol transport pathway of BmNPC2-BmNPC1,which synergistically mediates BmNPV infection with BmNPC1 protein,in which BmNPC1 protein plays a more important role.Cholesterol affects virus binding,membrane fusion,and virus budding.The results of this paper further deepen our understanding of the mechanism of baculovirus infection,provide new evidence for encapsulated virus hijacking cholesterol transport pathway to infect host cells,provide new ideas for cultivating BmNPV-resistant silkworm varieties for in-depth research on the interaction between important host factors and viruses.