Abstract

Bombyx mori nucleopolyhedrovirus (BmNPV) is a member of the Alphabaculovirus. Its major envelope glycoprotein, GP64, as a class III viral membrane fusion protein, plays critical roles in both viral entry and budding. GP64 must assemble into stable homotrimers to execute its functions. In this study, we demonstrate that the BmNPV GP64 homotrimer is stabilized by an intermolecular disulfide bond between Cys40 and Cys388. We employed site-directed mutagenesis to generate single (C40A, C388A) and double (C40A-C388A) cysteine-to-alanine substitution mutants and then investigated GP64 expression, oligomerization, membrane fusion activity, and viral infectivity. Our results show that the intermolecular disulfide bond in the BmNPV GP64 homotrimer is indispensable for its trimerization, membrane fusion capability, and the infectivity of budded virions (BV). This study not only deepens our understanding of the structural and functional roles of disulfide bonds in viral fusion proteins but also provides new experimental data for elucidating the infection mechanism of BmNPV.