Unveiling non-classical glycosylation patterns in Bombyx mori nucleopolyhedrovirus GP64: Insights into viral entry and fusion

Ying Xu¹, Yufeng Hao¹, Tingting Zhou², Stephen Baffour Gyawu¹, Luping Sun¹, Shijia Pan¹, Meixian Wang², Yujie Lu³, Bifang Hao², Jinshan Huang⁴

Affiliations

¹ Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China.
² Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China; Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang, 212100, China.
³ Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China; School of Grain Sciences and Technology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China.
⁴ Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, 212100, China; Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture and Rural Affairs, Sericultural Scientific Research Center, Chinese Academy of Agricultural Sciences, Zhenjiang, 212100, China. Electronic address: jshuang@just.edu.cn.

PMID: 38905921
DOI: 10.1016/j.virol.2024.110147

Abstract

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, SP^ΔnGP64, 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 SP^ΔnGP64. 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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