The Bombyx mori Nucleopolyhedrovirus GP64 Retains the Transmembrane Helix of Signal Peptide to Contribute to Secretion across the Cytomembrane.

Bombyx mori nucleopolyhedrovirus (BmNPV) is the primary pathogen of silkworms that causes severe economic losses in sericulture. GP64 is the key membrane fusion protein that mediates budded virus (BV) fusion with the host cell membrane. Previously, we found that the n-region of the GP64 signal peptide (SP) is required for protein secretion and viral pathogenicity; however, our understanding of BmNPV GP64 remains limited. Here, we first reported that BmNPV GP64 retained its SP in the mature protein and virion in only host cells but did not retain in nonhost cells. Uncleaved SP mediates protein targeting to the cytomembrane or secretion in Bombyx mori cells. The exitance of the n-region extended the transmembrane helix length, which resulted in the cleavage site to be located in the helix structure and thus blocked cleavage from signal peptidase (SPase). Without the n-region, the protein fails to be transported to the cytomembrane, but this failure can be rescued by the cleavage site mutation of SP. Helix-breaking mutations in SP abolished protein targeting to the cytomembrane and secretion. Our results revealed a previously unrecognized mechanism by which SP of membrane fusion not only determines protein localization but also determines viral pathogenicity, which highlights the escape mechanism of SP from the cleavage by SPase. IMPORTANCE BmNPV is the primary pathogen of silkworms, which causes severe economic losses in sericulture. BmNPV and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) are closely related group I alphabaculoviruses, but they exhibit nonoverlapping host specificity. Recent studies suppose that GP64 is a determinant of host range, while knowledge remains limited. In this study, we revealed that BmNPV GP64 retained its SP in host cells but not in nonhost cells, and the SP retention is required for GP64 secretion across the cytomembrane. This is the first report that a type I membrane fusion protein retained its SP in mature proteins and virions. Our results unveil the mechanism by which SP GP64 escapes cleavage and the role of SP in protein targeting. This study will help elucidate an important mechanistic understanding of BmNPV infection and host range specificity.

Methyl-Beta-Cyclodextrin-Induced Macropinocytosis Results in Increased Infection of Sf21 Cells by Bombyx Mori Nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) is closely related to Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with over ~93% amino acid sequence identity. However, their host ranges are essentially nonoverlapping. The mechanism of BmNPV entry into host cells is completely different from that of AcMNPV, and whether the entry mechanism difference relates to the host range remains unclear. BmNPV produces an abortive infection in nonhost cells due to virion nuclear transportation failure. Here, we performed a detailed study by increasing BmNPV infection in Sf21 cells with the aid of methyl-beta-cyclodextrin (MßCD). We found that low-concentration MßCD incubation efficiently activates membrane ruffling in Sf21 cells, which mediates the increase in BmNPV infection. Interestingly, MßCD incubation after virion internalization also increases the infection, which suggests that macropinocytosis is involved in BmNPV infection in Sf21 cells after virion internalization. Further study revealed that clathrin-mediated endocytosis (CME) is employed by BmNPV to facilitate entry into Sf21 cells, and chlorpromazine application abolishes BmNPV infection in cells incubated both with and without MßCD. Based on these studies, we show that BmNPV enters Sf21 cells via CME and that parallel induction of macropinocytosis facilitates BmNPV infection in Sf21 cells. This study reveals the mechanism of BmNPV entry into Sf21 cells and provides clues for improving BmNPV infections in nonpermissive cells.