The microsporidian Nosema bombycis is an obligate intracellular parasite of Bombyx mori, that lost its intact tricarboxylic acid cycle and mitochondria during evolution but retained its intact glycolysis pathway. N. bombycis hexokinase (NbHK) is not only a rate-limiting enzyme of glycolysis but also a secretory protein. Indirect immunofluorescence assays and recombinant HK overexpressed in BmN cells showed that NbHK localized in the nucleus and cytoplasm of host cell during the meront stage. When N. bombycis matured, NbHK tended to concentrate at the nuclei of host cells. Furthermore, the transcriptional profile of NbHK implied it functioned during N. bombycis’ proliferation stages. A knock-down of NbHK effectively suppressed the proliferation of N. bombycis indicating that NbHK is an important protein for parasite to control its host.

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.

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...