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
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苜蓿银纹夜蛾多核多角体病毒 GP64 的结构域 IV 内的关键残基和接触有助于其在膜融合过程中的重折叠
DOI：
10.1128/jvi.01105-20
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发表时间：
2020
影响因子：
5.4
通讯作者：
Li Zhaofei
中科院分区：
文献类型：
--
作者：
Yu Qianlong;Bai Lisha;Ji Ning;Yue Xiaorong;Jiang Yuanyuan;Li Zhaofei
苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）GP 64是一种III类病毒融合蛋白，其在病毒进入期间介导低pH触发的膜融合。虽然已经解决了融合后构象的GP 64的结构，但其融合前结构和蛋白质如何重折叠以执行融合的机制尚不清楚。在其融合后结构中，GP 64由五个结构域（结构域I至V）组成。结构域IV（氨基酸[aa] 374至407）含有两个环（环1和环2），其在分子的膜远端形成疏水口袋。为了确定结构域IV的作用，我们使用丙氨酸扫描诱变，以取代每个单独的残基和结构域IV内的接触形成残基，并评估其对GP 64介导的膜融合和病毒感染的贡献。在许多情况下，单个氨基酸的替换对GP 64没有显著影响。然而，R392的替换或N381-N385、N384-Y388、N385-W393或K389-W393接触的破坏导致修饰的GP 64的差的细胞表面表达和融合损失，而E390或G391的替换或N381-K389、N381-Q401、N381-Q401或K389-W393接触的破坏导致修饰的GP 64的差的细胞表面表达和融合损失。或N381-I403接触降低了构建体的细胞表面表达水平和GP 64介导融合孔扩张的能力。相反，N407的替换或D404-S406接触的破坏似乎限制融合孔扩张而不影响表达。结合这些结构体保持在融合前构象或在酸性条件下从融合前状态到融合后状态的转换效率显著降低的发现，我们提出结构域IV是膜融合过程中GP 64重折叠所必需的。重要信息杆状病毒GP 64与弹状病毒G、疱疹病毒gB和thogotovirus糖蛋白一起被归类为III类病毒融合蛋白。在它们的融合后结构中，这些蛋白质含有五个结构域（结构域I至V）。与弹状病毒G和疱疹病毒gB蛋白的结构域IV（由β折叠组成）不同，GP 64的结构域IV是环区;在thogotovirus糖蛋白中的相同结构域尚未解决。此外，结构域IV是接近域I（融合结构域）的水泡性口炎病毒（VSV）G和人巨细胞病毒（HCMV）gB的融合前结构，但在融合后的构象驻留在域I-远端的分子。在这项研究中，我们确定了高度保守的残基和接触AcMNPV GP 64的结构域IV内的低pH触发的构象变化和融合孔扩张是必要的。我们的研究结果突出了III类病毒融合蛋白的结构域IV在膜融合过程中重折叠的作用。
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 its postfusion structure, GP64 is composed of five domains (domains I to V). Domain IV (amino acids [aa] 374 to 407) 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 replace each of the individual residues and the contact-forming residues within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, replacement of a single amino acid had no significant impact on GP64. However, replacement of R392 or disruption of the N381-N385, N384-Y388, N385-W393, or K389-W393 contact resulted in poor cell surface expression and fusion loss of the modified GP64, whereas replacement of E390 or G391 or disruption of the N381-K389, N381-Q401, or N381-I403 contact reduced the cell surface expression level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, replacement of N407 or disruption of contact D404-S406 appeared to restrict fusion pore expansion without affecting expression. Combined with the finding that these constructs remain in the prefusion conformation or have a dramatically less efficient transition from the prefusion to the postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion.IMPORTANCEBaculovirus GP64 is grouped with rhabdovirus G, herpesvirus gB, and thogotovirus glycoproteins as a class III viral fusion protein. In their postfusion structures, these proteins contain five domains (domains I to V). Distinct from domain IV of rhabdovirus G and herpesvirus gB proteins, which is composed of β-sheets, domain IV of GP64 is a loop region; the same domain in thogotovirus glycoproteins 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 highly conserved residues and 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.