Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
J Biol Chem. 2021 Jan-Jun;296:100321. doi: 10.1016/j.jbc.2021.100321. Epub 2021 Jan 22.
During the late phase of HIV-1 infection, viral Gag polyproteins are targeted to the plasma membrane (PM) for assembly. Gag localization at the PM is a prerequisite for the incorporation of the envelope protein (Env) into budding particles. Gag assembly and Env incorporation are mediated by the N-terminal myristoylated matrix (MA) domain of Gag. Nonconservative mutations in the trimer interface of MA (A45E, T70R, and L75G) were found to impair Env incorporation and infectivity, leading to the hypothesis that MA trimerization is an obligatory step for Env incorporation. Conversely, Env incorporation can be rescued by a compensatory mutation in the MA trimer interface (Q63R). The impact of these MA mutations on the structure and trimerization properties of MA is not known. In this study, we employed NMR spectroscopy, X-ray crystallography, and sedimentation techniques to characterize the structure and trimerization properties of HIV-1 MA A45E, Q63R, T70R, and L75G mutant proteins. NMR data revealed that these point mutations did not alter the overall structure and folding of MA but caused minor structural perturbations in the trimer interface. Analytical ultracentrifugation data indicated that mutations had a minimal effect on the MA monomer-trimer equilibrium. The high-resolution X-ray structure of the unmyristoylated MA Q63R protein revealed hydrogen bonding between the side chains of adjacent Arg-63 and Ser-67 on neighboring MA molecules, providing the first structural evidence for an additional intermolecular interaction in the trimer interface. These findings advance our knowledge of the interplay of MA trimerization and Env incorporation into HIV-1 particles.
在 HIV-1 感染的晚期阶段，病毒 Gag 多聚蛋白被靶向到质膜（PM）进行组装。Gag 在 PM 的定位是将包膜蛋白（Env）纳入出芽颗粒的前提。Gag 组装和 Env 掺入由 Gag 的 N 端豆蔻酰化基质（MA）结构域介导。在 MA 的三聚体界面（A45E、T70R 和 L75G）发现非保守突变会损害 Env 的掺入和感染性，导致 MA 三聚化是 Env 掺入的必需步骤的假说。相反，MA 三聚体界面（Q63R）的补偿性突变可以挽救 Env 的掺入。这些 MA 突变对 MA 的结构和三聚体化特性的影响尚不清楚。在这项研究中，我们采用 NMR 光谱学、X 射线晶体学和沉降技术来表征 HIV-1 MA A45E、Q63R、T70R 和 L75G 突变蛋白的结构和三聚体化特性。NMR 数据表明这些点突变没有改变 MA 的整体结构和折叠，但在三聚体界面引起了微小的结构扰动。分析超速离心数据表明突变对 MA 单体-三聚体平衡的影响很小。未豆蔻酰化 MA Q63R 蛋白的高分辨率 X 射线结构揭示了相邻 MA 分子上侧链 Arg-63 和 Ser-67 之间的氢键，为三聚体界面中额外的分子间相互作用提供了第一个结构证据。这些发现增进了我们对 MA 三聚化和 HIV-1 颗粒中 Env 掺入相互作用的认识。
