Analysis Of Cholesterol-binding Site Activity In GP64 Of BmNPV Membrane Fusion Protein

Baculovirus is a type of DNA virus with a genome size of 80-180 kb that mainly infects Lepidoptera insects.Because it is harmless to people and the environment,it is widely used in production and life.In recent years,more studies have been made on Bombyx mori nucleopolyhedrovirus(BmNPV)and Autographa californica nucleopolyhedrovirus(AcMNPV),and the difference of their application in agriculture has aroused widespread concern.Although both are harmless to the environment,AcMNPV host domain is narrow but easy to obtain,while the host domain of BmNPV is narrow but is difficult to raise.If we combine the advantages of them,it will bring great economic benefits to agricultural production.In general,the envelope virus starts infection when the virus envelope glycoprotein specifically binds to cell surface receptors,and the receptor determines the host domain of the virus.The nucleopolyhedrovirus mainly uses GP64 as the budding virus.Therefore,the study of GP64 membrane fusion protein has far-reaching significance in exploring the invasion mechanism of the virus.BmNPV gp64 and AcMNPV gp64 are more than 95% homologous.The BmNPV envelope protein GP64 mediates virus entry into the cell by endocytosis of micropinocytosis.At the same time,our previous laboratory studies confirmed that cholesterol is also required for the invasion of BmNPV and is dose-dependent.Therefore,the mechanism of BmNPV invasion by cholesterol is not clear.This article explores the relationship between BmNPV gp64 cholesterol recognition sites and virus invading host cells from the following three aspects:1.Construction of BmNPV gp64 Deletion Virus: Deletion primers with homology arms were designed to delete the BmNPV gp64 gene by homologous recombination.2.BmNPV gp64 CRACs infectivity verification: There are four cholesterol recognition sites on BmNPV gp64,and their key amino acid Y mutations are restored to BmNPV gp64 deletion Bacmids.The relationship between four cholesterol recognition sites and virus infectivity is explored.3.BmNPV GP64 protein expression and biological activity assay: The effect of point mutations on the expression of GP64 protein was analyzed by Western-Blot,virus rescue,and immunofluorescence.The effect of point mutation gp64 on the biological activity of the virus was determined by plotting the growth curve and median lethal time.Through research we preliminarily revealed the main mechanism of BmNPV invasion of host cells.BmNPV cholesterol recognition sequences CRAC2,CRAC3 are involved in amplification of the virus.