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

Abstract

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.

Label-free proteomics analysis on the envelope of budded viruses of Bombyx mori nucleopolyhedrovirus harboring differential localized GP64

“…Our previous studies revealed that BmNPV BV infection was different from that of AcMNPV, including direct membrane fusion that caused nucleocapsid transportation failure to the nucleus [20], the exact cause that BmNPV failure to infect nonhost cells [9]. BmNPV GP64 retained its SP in the mature virion [10], and this uncleaved SP determined BmNPV's special dependence on cholesterol in the virus infection process [11,21]. Viral proteins and host proteins in the envelope play important roles in the BV infection process, hereby, proteomics composition analysis of the BV envelope will facilitate the understanding of BmNPV budding and infection process.…”

“…BmN cells were cultured at 27 ℃ using standard techniques in TC-100 insect medium (AppliChem, Darmstadt, Germany) supplemented with 10% FBS (Gibco BRL, Gaithersburg, MD, USA). BmBac Δgp64 -gp64 (vGP64) and BmBac Δgp64 -SP ∆n gp64 (vSP ∆n GP64) were previously constructed by Hao et al [10,11]. In brief, BmNPV gp64 gene in BmBacmid was replaced with chloramphenicol to generate gp64-null bacmid (BmBac Δgp64 ) [12], then wildtype gp64 was reintroduced into the polyhedrin locus of BmBac Δgp64 by transposition to generate BmBac Δgp64 -gp64 [10].…”

“…BmBac Δgp64 -gp64 (vGP64) and BmBac Δgp64 -SP ∆n gp64 (vSP ∆n GP64) were previously constructed by Hao et al [10,11]. In brief, BmNPV gp64 gene in BmBacmid was replaced with chloramphenicol to generate gp64-null bacmid (BmBac Δgp64 ) [12], then wildtype gp64 was reintroduced into the polyhedrin locus of BmBac Δgp64 by transposition to generate BmBac Δgp64 -gp64 [10]. Meanwhile, an SP n-region null-gp64 (18 amino acid was deleted in the n-terminal of wild-type GP64, shown in Fig.…”

“…Meanwhile, an SP n-region null-gp64 (18 amino acid was deleted in the n-terminal of wild-type GP64, shown in Fig. 1A) was also reintroduced into BmBac Δgp64 to generate BmBac Δgp64 -SP ∆n gp64 [10], n-region absence caused SP ∆n GP64 intracellular localization and weak infectivity [10,11]. BmN cells in a flask (75 cm 2 , NEST Biotechnology, Wuxi, China) were infected with the viruses at a multiplicity of infection (MOI) of 5 TCID 50 per cell, and the supernatants were collected from the infected BmN cells at 72 h post-infection (h p.i.).…”

“…In our previous studies, we revealed that the BmNPV GP64 signal peptide (SP) was retained in mature BV, BmNPV GP64 contained a typical SP, which is composed of n-, h-, and c-region, the longer n-region caused GP64 SP to escape the cleavage from SPase and thus mediated GP64 PM localization [10], and the fact that SP-cleaved GP64 (SP ∆n GP64) failed to target PM but was assembled into BV is distinct from the current budding theory that BV acquired its envelope from the PM [11]. Moreover, Ebola virus glycoprotein was efficiently expressed in BmN cells but failed to be transported to PM [12], this non-PM localized glycoprotein can also be incorporated into BV [13].…”