In the acidic pH environment of endosomes, GP64 undergoes conformational changes to expose certain domain(s) for better access by BmNPC1. decrease viral entry (2 hpi) rather than reduce the viral binding to the BmE cells. Furthermore, our results showed that NPC1 domain C binds directly and specifically to the viral glycoprotein GP64, which is responsible for both receptor binding and fusion. Antibody blocking assay also revealed that the domain C specific polyclonal antibody inhibited CPI 0610 BmNPV infection, indicating that NPC1 domain C most likely plays a role during viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of human NPC1 (hNPC1) in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of exploiting host NPC1 proteins during virus intracellular CPI 0610 entry events. receptor expression-enhancing protein (BmREEPa) (Dong et al., 2015) have been identified to be involved in BV attachment or binding, the exact identity of host receptors for baculovirus still remains elusive. Among numerous baculoviruses, nuclear polyhedrosis virus (BmNPV) is one of the most frequently employed in the research, which showed close relatedness with multiple nucleopolyhedrovirus (AcMNPV). It has been reported previously that BmNPV infection was enhanced by promoting protein (BmPP), a family member of Niemann-Pick C2 (NPC2), in silkworm BoMo cells (Kanaya and Kobayashi, 2000). The addition of BmPP to culture media at a concentration of 1 1 g/ml resulted in a 1000C10,000-fold increase of BmNPV production; however, the detailed molecular mechanism has not yet been fully elucidated. Human NPC2 protein functions as a central shuttle binding to Niemann-Pick C1 (NPC1) to export low density lipoprotein (LDL)-derived cholesterol from late endosomes and lysosomes to other cellular compartments (Infante et al., 2008). The role of BmPP in promoting BmNPV infection indicates that host proteins responsible for cholesterol transport may be involved in baculovirus infection. Interestingly, the infection of Ebola virus (EBOV) has proven to be dependent on cholesterol transporter NPC1, which serves as a virus intracellular receptor for filovirus entry (Carette et al., 2011; C?t et al., 2011; Miller et al., 2012). NPC1 is a ubiquitously expressed membrane protein Sema4f which is mainly involved in intracellular cholesterol transport (Altmann et al., 2004). Structurally, NPC1 is comprised of three large luminal loop domains, A, C, and I (Carstea et al., CPI 0610 1997; Loftus et al., 1997). Domain A, also called the N-terminal domain (NTD), is involved in cholesterol binding and exporting cholesterol from lysosomes into the cytosol. Domain C directly and specifically binds to NPC2 and constitutes the scaffold to properly accommodate NPC2 for hydrophobic handoff of cholesterol to the pocket of NTD (Infante et al., 2008; Deffieu and Pfeffer, 2011; Gong et al., 2016). Furthermore, NPC1 domain C is able to bind to the cathepsin-primed form of Ebola glycoprotein (GPcl) (Gong et al., 2016; Han et al., 2016); the absence of domain C results in complete resistance to infection by EBOV, indicating that this domain is essential for EBOV entry. The third luminal domain I interacts with domain C, and may play a supporting role in cholesterol transport (Gong et al., 2016). The recent findings of NPC1 protein as an intracellular receptor for filovirus, coupled with prior evidence supporting the involvement of NPC2 (BmPP) in BmNPV infection, prompted us to investigate the potential role of NPC1 homologs in BmNPV infection in insect cells. Here, we demonstrate that BV of BmNPV infection requires the expression of BmNPC1. BmE cells pre-treated with the NPC1 inhibitors imipramine or U18666A, which can CPI 0610 mimic the molecular phenotype of NPC disease (Underwood et al., 1996; Jupatanakul et al., 2014; Wichit et al., 2017), results in marked inhibition of viral replication and production. Silencing, or knocking out of BmNPC1 expression also substantially impairs BmNPV proliferation cell line BmE cells were maintained at 28C in Graces medium (Thermo Fisher Scientific, United States) supplemented with 10% (V/V) fetal bovine serum (FBS) (Thermo Fisher Scientific, United States) and 1%(V/V) penicillin-streptomycin (Pan et al., 2010). The recombinant BmNPV virus bearing an EGFP gene under the control of polyhedrin promoter was constructed by Bac-to-Bac Baculovirus Expression System according to the manufacturers protocol (Invitrogen, United States). The expression of EGFP can act as a reporter for monitoring viral CPI 0610 gene expression and viral replication (Zhang et al., 2014). Recombinant viruses were propagated in BmE cells and viral titers were measured by 50% tissue culture infectious dose (TCID50) based on EGFP expression as described (Wu et al., 2010). Cloning, Protein Expression,.