Abstract
Bombyx mori cypovirus (BmCPV), a member of the Reoviridae, specifically infects silkworms and causes extensive economic losses to the sericulture industry. To date, the entry mechanism of BmCPV into cells is unclear. Here we used electron microscopy to study the route of entry of BmCPV into cells, and the results demonstrated that the entry of BmCPV into BmN cells was mediated by endocytosis. Blocking the entry pathway with four endocytosis inhibitors, including dansylcadaverine, chlorpromazine, genistein, and PP2, significantly decreased the infectivity of BmCPV. This indicates that BmCPV enters BmN cells via endocytosis, and that clathrin-mediated sorting is the predominant entry method. After the relative expression levels of clathrin heavy chain (clathrin, GenBank accession No. NM_001142971.1) and the adaptor protein complex-1 gamma subunit AP-1 (AP-1, GenBank accession No. JQ824201.1), which are involved in clathrin-mediated endocytosis, were inhibited by RNA interference or abolishing the functions of clathrin and AP-1 with their corresponding antibodies, the infectivity of BmCPV was reduced significantly, which suggests that clathrin-mediated endocytosis contributed to the entry of BmCPV into cells. Our findings suggest that the clathrin-mediated endocytosis pathway is a candidate for the development of therapeutics for silkworm cytoplasmic polyhedrosis.
Highlights
Cytoplasmic polyhedrosis viruses (CPVs) are the second major group of insect pathogens belonging to the genus Cypovirus in the family Reoviridae[1]
To further understand the functions of endocytosis during the cell entry of Bombyx mori cypovirus (BmCPV), we investigated the effects of chlorpromazine, dansylcadaverine, genistein, and PP2 which were the endocytic inhibitors on the entry of BmCPV into BmN cells
We used confocal microscopy to further examine the effect of these four inhibitors on the internalization of BmCPV into BmN cells, the results showed that the number of BmCPV virions in the BmN cells treated with inhibitors was less than that in control cells (Fig. 3a–e), suggesting that the entry of BmCPV into BmN cells was inhibited by endocytic inhibitors
Summary
Cytoplasmic polyhedrosis viruses (CPVs) are the second major group of insect pathogens belonging to the genus Cypovirus in the family Reoviridae[1]. The nonstructural proteins (NSPs) p101 (NSP5), p44 (NSP8), NS5 (NSP9), and polyhedrin are encoded by the S5, S8, S9 and S10 genes, respectively Among these proteins, VP3, a spike protein, was predicted to bind to a receptor located on the surface of host cells during the CPV infection process[6]. Rice dwarf virus (RDV) is a member of the genus Phytoreovirus in the family Reoviridae, and the entry of RDV into Nephotettix cincticeps cells was demonstrated to depend on clathrin-mediated endocytosis, which is true for mammalian reoviruses[13], suggesting that endocytosis plays an important role in the early events of reovirus infection. It has been proven that Src kinase can regulate the proper sorting of virus particles in the endocytosis pathway, and that it helps disassemble viruses, which promotes viral cell entry. There is no silkworm variety that is highly resistant to BmCPV; protecting silkworms from BmCPV infection is conducted by inactivating BmCPV virions that exist in the rearing environment using disinfectors, and by enhancing the resistance of silkworms through feeding and management during cocoon production; the prevention and control of silkworm cytoplasmic polyhedrosis in sericulture remains a large problem
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