Qianlong Yu, Lisha Bai, Ning Ji, Xiaorong Yue, Yuanyuan Jiang, Zhaofei Li.Critical residues and contacts within domain IV of Autographa californica multiple nucleopolyhedrovirus GP64 contribute to its refolding during membrane fusion

Critical residues and contacts within domain IV of Autographa californica multiple nucleopolyhedrovirus GP64 contribute to its refolding during membrane fusion

Qianlong Yu, Lisha Bai, Ning Ji, Xiaorong Yue, Yuanyuan Jiang, Zhaofei Li

Journal of Virology

DOI: 10.1128/JVI.01105-20

Abstract

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In postfusion structure, GP64 is composed of five domains (domain I-V). Domain IV (374-407 aa) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to substitute each of the residues and the contacts within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, substitution of a single amino acid has no significant impact on GP64. However, substitution of R392 or disrupting the contact N381-N385, N384-Y388, N385-W393, or K389-W393 resulted in poor cell surface expression and fusion loss of the modified GP64, whereas substitution of E390 or G391, or disrupting the contact N381-K389, N381-Q401, or N381-I403 reduced the cell surface level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, substitution of N407 or disrupting the contact D404-S406 appears to restrict fusion pore expansion without affecting expression. Combined with the identification of these constructs remaining stable prefusion conformation or dramatically less efficient transition from a prefusion to postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion.Importance Baculoviruses GP64 is grouped with rhabdoviruses G, herpesviruses gB, and thogotoviruses glycoproteins as class III viral fusion proteins. In their postfusion structures, these proteins contain five domains (domain I-V). Distinguished from domain IV of rhabdoviruses G and herpesviruses gB that composed of β-sheets, domain IV of GP64 is a loop region and the same domain in thogotovirues glycorproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that the highly conserved residues and the contacts within domain IV of AcMNPV GP64 are necessary for low-pH triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.