The glycoprotein GP64 of alphabaculovirus is crucial for viral entry and fusion. Here, we investigated the N-glycosylation patterns of Bombyx mori nucleopolyhedrovirus (BmNPV) GP64 and its signal peptide (SP) cleaved form, SPΔnGP64, along with their impacts on viral infectivity and fusogenicity. Through deglycosylation assays, we confirmed N-glycosylation of BmNPV GP64 on multiple sites. Mutational analysis targeting predicted N-glycosylation sites revealed diverse effects on viral infectivity and cell fusion. Particularly noteworthy were mutations at sites 175, which resulted in complete loss of infectivity and fusion capacity. Furthermore, LC-MS/MS analysis uncovered unexpected non-classical N-glycosylation sites, including N252, N302, N367, and N471, with only N302 and N471 identified in SPΔnGP64. Subsequent investigation highlighted the critical roles of these residues in BmNPV amplification and fusion, underscoring the essentiality of N367 glycosylation for GP64 fusogenicity. Our findings provide valuable insights into the non-classical glycosylation landscape of BmNPV GP64 and its functional significance in viral biology.
Keywords: Baculovirus; GP64; Membrane fusion; N-Glycosylation; Non-classical pattern; Signal peptide.
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