Liang Yu, Guttman Miklos, Williams James A, Verkerke Hans, Alvarado Daniel, Hu Shiu-Lok, Lee Kelly K
Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.
Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.
J Virol. 2016 Sep 29;90(20):9224-36. doi: 10.1128/JVI.01116-16. Print 2016 Oct 15.
The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines.
The HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.
包膜糖蛋白（Env）是HIV-1广泛中和抗体（bNAbs）的主要靶点。HIV进化出逃避宿主免疫反应的机制之一是用密集的糖基化掩盖Env上的保守表位。先前的研究表明，去除N197处的特定保守聚糖会增加病毒对靶向CD4结合位点（CD4bs）抗体的中和敏感性，从疫苗设计的角度来看，这使其成为一个备受关注的位点。目前，去除N197聚糖所导致的结构后果尚未得到表征。我们使用类天然的SOSIP三聚体，研究去除该聚糖对抗原性和局部结构动力学的影响。在三聚体Env中观察到N197Q突变体与CD4bs和靶向V3的抗体的结合大幅增加，而单体gp120则未观察到变化。虽然整体结构和热稳定性未改变，但通过氢氘交换质谱法在N197Q突变体中明显观察到相邻原体三聚体界面处可变环的灵活性略有增加。聚糖链的结构建模表明，N197聚糖的空间占据导致Env三聚体中与CD4bs抗体发生空间冲突，但单体gp120中则不会。我们的结果表明，去除N197聚糖可增强Env上相关bNAb表位的暴露，同时对整体三聚体结构的影响最小。这些发现为增强疫苗中三聚体Env免疫原提供了一种简单的修饰方法。
HIV-1 Env糖蛋白呈现出由宿主细胞衍生的N-连接聚糖组成的密集拼图。这种所谓的聚糖屏蔽被认为是一种主要的免疫识别保护机制。虽然许多N-连接聚糖的位置具有毒株特异性，但有些是高度保守的，并且被认为发挥关键的功能作用。在本研究中，我们研究了保守的、靠近CD4结合位点的N197聚糖，并证明其去除既有助于中和抗体接近CD4结合位点，又适度影响三聚体冠部的结构动力学，而不会大幅改变整体Env三聚体稳定性。这表明手术性糖基化位点修饰可能是塑造基于Env的疫苗中表位呈现的有效方法。
