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Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

ABSTRACT: Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP1,2 expression in BmN cells, EBOV-GP1,2 with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid via transposition. The infectivity of the recombinant bacmids was investigated after transfection, expression and localization of EBOV-GP were investigated, and cell morphological changes were analyzed by TEM. The GP64 signal peptide, but not the GP1,2 native signal peptide, caused GP1,2 localization to the cell membrane, and the differentially localized GP1,2 proteins were cleaved into GP1 and GP2 fragments in BmN cells. GP1,2 expression resulted in dramatic morphological changes in BmN cells in the early stage of infection. However, GP1,2 expression did not rescue GP64 deficiency in BmNPV infection. This study provides a better understanding of GP expression and processing in BmN cells, which may lay a foundation for EBOV-GP expression using the BmNPV baculovirus expression system.

SUBMITTER: Huang J

PROVIDER: S-EPMC6893839 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

Huang Jinshan J Liu Na N Xu Fanbo F Ayepa Ellen E Amanze Charles C Sun Luping L Shen Yaqin Y Yang Miao M Yang Shuwen S Shen Xingjia X Hao Bifang B

Viruses 20191115 11

Ebola virus (EBOV) disease outbreaks have resulted in many fatalities, yet no licensed vaccines are available to prevent infection. Recombinant glycoprotein (GP) production may contribute to finding a cure for Ebola virus disease, which is the key candidate protein for vaccine preparation. To explore GP<sub>1,2</sub> expression in BmN cells, EBOV-GP<sub>1,2</sub> with its native signal peptide or the GP64 signal peptide was cloned and transferred into a normal or gp64 null Bombyx mori nucleopoly ...[more]

PMID: 31731691

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Project description: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.

Dataset Information

Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

Publications

Efficient Expression and Processing of Ebola Virus Glycoprotein Induces Morphological Changes in BmN Cells but Cannot Rescue Deficiency of Bombyx Mori Nucleopolyhedrovirus GP64.

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