The NPC Families Mediate BmNPV Entry

Baculovirus is a powerful tool for biological control in agriculture and foreign gene expression and delivery in insect and mammalian cells. Baculovirus enters host cells by multiple endocytic pathways; however, the current understanding of the Bombyx mori nucleopolyhedrovirus (BmNPV) entry mechanism remains limited. Previous studies have identified NPC1 and NPC2 as important host factors for viral infection in insect cells, although their exact role in viral infection has not yet been determined. In this study, we demonstrate that the BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 in Sf9 cells confers the virus the ability to enter into the nucleus of Sf9 cells. Additionally, the second luminal domain of BmNPC1 (BmNPC1-C) binds to the viral glycoprotein gp64, and preincubation of BmNPV with purified BmNPC1-C inhibits virus infection. Furthermore, knockout of the BmNPC2 protein results in reduced efficiency of viral fusion with the endosomal membrane, and BmNPC2 protein interacts directly with both viral envelope glycoprotein gp64 and the host BmNPC1 protein. BmNPC2 was found to be incorporated into progeny viral particles. Taken together, our results suggest that NPC2 protein incorporated into viral particles may facilitate viral infection through promoting the interaction of BmNPV and NPC1 in the endosome, thus enhancing viral fusion and escape from endosomes. These results, combined with those from previous studies, support that BmNPV hijacks two important cholesterol receptor members (NPC1 and NCP2) in the cholesterol intracellular transport pathway for viral entry into host cells. IMPORTANCE Baculovirus is an important biological factor for controlling insect populations and represents a powerful biological tool for gene delivery and expression. However, the host receptor of baculovirus is still unknown. In this study, we demonstrate that BmNPC1 protein is an important intracellular factor for BmNPV escape from the endosomal compartment, and the expression of BmNPC1 confers the ability of virus to enter into the host cell nucleus in nonpermissive Sf9 cells. BmNPC2 can bind to the virus and promote progeny virion infection through the NPC1-NPC2 endosome cholesterol transport pathway. We believe that our study on the BmNPV entry mechanism will further facilitate the application of baculovirus systems in eukaryotic gene delivery. Not only can the cholesterol transport pathway NPC1 protein be used by a variety of enveloped viruses, but the NPC2 protein can also be used by viruses to infect host cells. This will provide new insights into the study of enveloped virus infection mechanisms.