Both ganglioside GM2 and cholesterol in the cell membrane are essential for Bombyx mori cypovirus cell entry

Bombyx mori cypovirus (BmCPV) enters permissive cells via clathrin-mediated endocytosis pathway. However, the distinct entry mechanism for BmCPV is still ambiguous. The aim of this study is to investigate the role of gangliosides and cholesterol in BmCPV cell entry. The number of BmCPV virions attached to the cell surface and the expression level of BmCPV vp1 gene was significantly decreased by digestion of terminal sialic acids in gangliosides with neuraminidase (NA). Preincubation of different concentration of ganglioside GM1, GM2 or GM3 with BmCPV prior to infection, the reduction of BmCPV infectivity was found by GM2-treated in a dose-depend manner. BmCPV virions were found to colocalize with GM2 in the cell surface. The infectivity of BmCPV was reduced by anti-GM2 antibody treatment cells. Moreover, BmCPV infection was impaired by depletion of membrane cholesterol with MβCD, but the inhibitory effect of MβCD was restored by supplementing with cholesterol. The number of viral particles attached on the BmN cells was significantly decreased by pretreated with MβCD, and BmCPV infection was inhibited by silencing the expression of 3-hydroxy-3-methylglutaryl-CoA reductase gene (Hmg-r) in cholesterol biosynthesis pathway. These results indicate that ganglioside GM2 and cholesterol in membrane lipid rafts are essential for BmCPV attachment to cell surface for its cell entry.