The dual roles of baculovirus for the control of natural insect populations as an insecticide, and as a tool for foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for baculovirus infection in insect cells. Both pretreatment of B. mori embryonic cells (BmE) with NPC1 antagonists (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV (B. mori nuclear polyhedrosis virus) infectivity. Disruption of BmNPC1 could 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 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 entry events.

Background Microsporidians are opportunistic pathogens with a wide range of hosts, including invertebrates, vertebrates and even humans. Microsporidians possess a highly specialized invasion structure, the polar tube. When spores encounter an appropriate environmental stimulation, the polar tube rapidly everts out of the spore, forming a 50–500 µm hollow tube that serves as a conduit for sporoplasm passage into host cells. The polar tube is mainly composed of polar tube proteins (PTPs). So far, five major polar tube proteins have been isolated from microsporidians. Nosema bombycis, the first identified microsporidian, infects the economically important insect silkworm and causes heavy financial loss to the sericulture industry annually. Results A novel polar tube protein of N. bombycis (NbPTP6) was identified. NbPTP6 was rich in histidine (H) and serine (S), which contained a signal peptide of 16 amino acids at the N-terminus. NbPTP6 also had 6 potential O-glycosylation sites and 1 potential N-glycosylation site. The sequence alignment analysis revealed that NbPTP6 was homologous with uncharacterized proteins from other microsporidians (Encephalitozoon cuniculi, E. hellem and N. ceranae). Additionally, the NbPTP6 gene was expressed in mature N. bombycis spores. Indirect immunofluorescence analysis (IFA) result showed that NbPTP6 is localized on the whole polar tube of the germinated spores. Moreover, IFA, enzyme-linked immunosorbent (ELISA) and fluorescence-activated cell sorting (FACS) assays results revealed that NbPTP6 had cell-binding ability. Conclusions Based on our results, we have confirmed that NbPTP6 is a novel microsporidian polar tube protein. This protein could adhere with the host cell surface, so we speculated it might play an important role in the process of microsporidian infection.

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.

The dual roles of baculovirus for the control of natural insect populations as an insecticide, and for 12 foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms 13 governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for 14 baculovirus infection in insect cells. Both pretreatment of Bombyx mori embryonic cells (BmE) with NPC1 antagonists 15 (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus 16BmNPV (Bombyx mori nuclear polyhedrosis virus) infectivity. Furthermore, we show that the major glycoprotein gp64 17 of BmNPV, responsible for both receptor binding and fusion, is able to interact predominantly with the BmNPC1 C 18 domain, with an enhanced binding capacity at low pH conditions, indicating that NPC1 most likely plays a role during 19 viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of 20 hNPC1 in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of 21 exploiting host NPC1 proteins during virus intracellular entry events. 22 IMPORTANCE BmNPV is one of the most important members of the Baculoviridae; many viruses in this family 23 have been frequently employed as viral vectors for foreign gene delivery or expression and as biopesticides, but their 24 host receptors still remain unclear. Here, we describe that the intracellular cholesterol transporter BmNPC1 is 25 indispensable for BmNPV infection in insect cells, and it interacts with the major viral glycoprotein gp64. Our study on 26 the role of BmNPC1 in baculovirus infection has further expanded the list of the enveloped viruses that require host 27 NPC1 proteins for entry, and will ultimately help us to uncover the molecular mechanism of the involvement of NPC1 28 proteins in the entry process of many enveloped viruses. 29 KEYWORDS Baculovirus, NPC1, host factor, virus entry, cholesterol trafficking pathway, enveloped virus, 30 Ebola virus 31 32Enveloped viruses include a number of pathogens with significant importance to animal or human health. As a 33 typical double-stranded DNA enveloped virus, baculoviruses are known to infect invertebrates, with over 600 host 34 species described (1, 2). Due to a strong species-specific tropism for arthropods, baculovirus has been widely used as a 35 biopesticide (3). Concurrently, baculovirus can transduce a broad range of vertebrate cells, including human, bovine, 36 fish avian, and even primitive cells such as embryonic stem cells, warranting its application as a biological tool for gene 37 delivery (4-6). During its viral infection cycle, baculovirus produces two virion phenotypes: occlusion-derived virus 38 (ODV) which transmits to insects by the oral route and specializes to infect midgut epithelial cells, and budding virus 39 (BV) produced during nucleocapsid budding from the basolateral membrane of infected cells, respons...

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