Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
J Virol. 2019 May 15;93(11). doi: 10.1128/JVI.00219-19. Print 2019 Jun 1.
HIV-1 infection is initiated by viral Env engaging the host receptor CD4, triggering Env to transition from a "closed" to "open" conformation during the early events of virus-cell membrane fusion. To understand how Env sequence accommodates this conformational change, mutational landscapes decoupled from virus replication were determined for Env from BaL (clade B) and DU422 (clade C) isolates interacting with CD4 or antibody PG16 that preferentially recognizes closed trimers. Sequence features uniquely important to each bound state were identified, including glycosylation and binding sites. Notably, the Env apical domain and trimerization interface are under selective pressure for PG16 binding. Based on this key observation, mutations were found that increase presentation of quaternary epitopes associated with properly conformed trimers when Env is expressed at the plasma membrane. Many mutations reduce electrostatic repulsion at the Env apex and increase PG16 recognition of Env sequences from clades A and B. Other mutations increase hydrophobic packing at the gp120 inner-outer domain interface and were broadly applicable for engineering Env from diverse strains spanning tiers 1, 2, and 3 across clades A, B, C, and BC recombinants. Core mutations predicted to introduce steric strain in the open state show markedly reduced CD4 interactions. Finally, we demonstrate how our methodology can be adapted to interrogate interactions between membrane-associated Env and the matrix domain of Gag. These findings and methods may assist vaccine design. HIV-1 Env is dynamic and undergoes large conformational changes that drive fusion of virus and host cell membranes. Three Env proteins in a trimer contact each other at their apical tips to form a closed conformation that presents epitopes recognized by broadly neutralizing antibodies. The apical tips separate, among other changes, to form an open conformation that binds tightly to host receptors. Understanding how Env sequence facilitates these structural changes can inform the biophysical mechanism and aid immunogen design. Using deep mutational scans decoupled from virus replication, we report mutational landscapes for Env from two strains interacting with conformation-dependent binding proteins. Residues in the Env trimer interface and apical domains are preferentially conserved in the closed conformation, and conformational diversity is facilitated by electrostatic repulsion and an underpacked core between domains. Specific mutations are described that enhance presentation of the trimeric closed conformation across diverse HIV-1 strains.
HIV-1 感染是由病毒包膜蛋白（Env）与宿主受体 CD4 结合引发的，这一过程触发了 Env 在病毒与细胞膜融合的早期事件中从“封闭”构象向“开放”构象的转变。为了了解 Env 序列如何适应这种构象变化，我们对与 CD4 或优先识别封闭三聚体的抗体 PG16 相互作用的 BaL（B 型）和 DU422（C 型）分离株的 Env 进行了与病毒复制无关的突变景观分析。确定了与每个结合状态独特相关的序列特征，包括糖基化和结合位点。值得注意的是，Env 的尖顶结构域和三聚体化界面受到 PG16 结合的选择性压力。基于这一关键观察结果，发现了一些突变，这些突变增加了与适当构象三聚体相关的四级表位的呈现，当 Env 在质膜上表达时。许多突变减少了 Env 顶点的静电排斥，并增加了 PG16 对来自 A 型和 B 型谱系的 Env 序列的识别。其他突变增加了 gp120 内外结构域界面的疏水性堆积，并且可广泛应用于工程来自 A、B、C 和 BC 重组谱系的 1、2 和 3 层的不同菌株的 Env。预测在开放状态下引入空间应变的核心突变显示出明显降低的 CD4 相互作用。最后，我们展示了如何调整我们的方法来研究膜相关 Env 与 Gag 基质结构域之间的相互作用。这些发现和方法可能有助于疫苗设计。HIV-1 Env 是动态的，经历了很大的构象变化，从而驱动病毒和宿主细胞膜的融合。三聚体中的三个 Env 蛋白在其尖顶处相互接触，形成一个封闭的构象，该构象呈现被广泛中和抗体识别的表位。除其他变化外，尖顶部分离，形成一个与宿主受体紧密结合的开放构象。了解 Env 序列如何促进这些结构变化可以提供生物物理机制的信息，并有助于免疫原设计。使用与病毒复制分离的深度突变扫描，我们报告了与两种与构象依赖性结合蛋白相互作用的菌株的 Env 突变景观。在封闭构象中，Env 三聚体界面和尖顶结构域中的残基优先保守，而静电排斥和域之间的未填充核心促进了构象多样性。描述了特定的突变，这些突变增强了不同 HIV-1 株系的三聚体封闭构象的呈现。
