Defining the impact of glycan-microdomains of the HIV-1 Env glycan shield

摘要 人类免疫缺陷病毒1型(HIV-1)是全球主要病原体，2019年新增感染170万人。 HIV-1包膜糖蛋白(Env)是唯一的病毒表面抗原。Env，gp120/gp41二聚体的三聚体 亚基，由N-糖链密集糖基化而成。对于gp120亚基，N-糖链贡献了其50%以上的亚基。 分子质量，并形成与细胞受体相互作用的保护性表面，即所谓的“多聚糖屏障” 和免疫系统，保护病毒免受中和抗体(NAB)的侵袭。最近，一种 基于对免疫逃逸变异体的序列分析，有人提出了“进化中的糖壳”。 失去了一些潜在的N-糖基化位点(NGS)，获得了另一些。尽管在这方面取得了很大进展 对HIV-1env生物学的了解，在分子水平上对N-糖链的复杂性和功能的了解很少 明白了。基于我们的生物信息学和实验研究，我们最近提出了环境多糖 屏蔽可以被定义和分析为不同的结构糖微结构域。使用自然发生的环境 B分支病毒的变种，我们分析了高度保守的N262糖链周围的糖链，在SO-2中。 在环境三聚体顶端附近称为高甘露糖斑(HMP)。这些HMP多聚糖由不同的 在N262附近，4到6个NGS的组合似乎形成了一个多糖工作单位。基于我们的 已发表的结果和初步数据，我们假设环境外区，gp120葡聚糖盾，具有 可以被定义为不同的结构糖微结构域的结构成分。我们建议 在HIV-1的不同分支及其免疫逃逸变体中测试我们的假设，方法是评估结构和 组成环境多糖微域的一系列N-葡聚糖组合的功能影响。具体来说， 我们建议确定N262-锚定的HMP-微域的不同NGS组合如何跨越 变体和分支影响环境功能(目标1)，以探索不同的N262-葡聚糖的结构排列 在功能性重组环境三聚体(目标2)的背景下对NGS组合进行聚类，并评估 HIV-1包膜糖链的其他潜在糖簇的功能影响(目标3)。要实现这些目标 目标，我们将使用功能、生物分析、结构生物信息学和分子动力学的组合 模拟方法。这些研究的结果将检验我们提出的关于功能影响的假设 多聚糖的结构成分屏蔽了多聚糖-微域，并将决定其如何变化 这些微域的组成影响着糖壳的功能。这些数据将推动我们的 了解HIV-1的分子生物学以及影响病毒传播和持久性的因素。 最终，这些研究将生成有关环境漏洞的新信息，可利用这些信息进行开发 未来的治疗方法。

ABSTRACT Human immunodeficiency virus 1 (HIV-1) is a major global pathogen, with 1.7 million new infections in 2019. The HIV-1 envelope glycoprotein (Env) is the sole virus-surface antigen. Env, a trimer of gp120/gp41 dimer subunits, is densely glycosylated by N-glycans. For the gp120 subunit, N-glycans contribute over 50% of its molecular mass and form a protective surface, the so-called “glycan shield”, that interacts with cellular receptors and the immune system and protects the virus from neutralizing antibodies (nAbs). Recently, the concept of an “evolving glycan shield” has been proposed, based on the sequence analysis of immune-escape variants that have lost some potential N-glycosylation sites (NGS) and gained others. Despite significant strides in understanding the biology of HIV-1 Env, the complexity and function of N-glycans at a molecular level are poorly understood. Based on our bioinformatics and experimental studies, we recently proposed that the Env glycan shield may be defined, and analyzed, as distinct structural glycan-microdomains. Using naturally occurring Env variants of a clade B virus, we analyzed the glycans surrounding the highly conserved N262 glycan, in the so- called high-mannose patch (HMP) near the apex of the Env trimer. These HMP glycans, comprised of different combinations of 4 to 6 NGS in the proximity of N262, appear to form a glycan working unit. Based on our published results and preliminary data, we hypothesize that Env outer domain, gp120 glycan shield, has structural components that can be defined as distinct structural glycan-microdomains. We propose to test our hypothesis in different clades of HIV-1 and their immune-escape variants by assessing structural and functional impacts of a range of N-glycan combinations that comprise Env glycan-microdomains. Specifically, we propose to determine how different NGS combinations of the N262-anchored HMP-microdomain across variants and clades influence Env functions (Aim 1), to probe the structural arrangement of different N262-glycan cluster NGS combinations in the context of functional recombinant Env trimers (Aim 2), and to assess the functional impacts of other potential glycan-clusters of the HIV-1 Env glycan shield (Aim 3). To accomplish these goals, we will use a combination of functional, bioanalytical, structural bioinformatics, and molecular dynamics simulation approaches. The results of these studies will test our proposed hypothesis on the functional impact of the structural components of the glycan shield, glycan-microdomains, and will determine how the varied compositions of these microdomains affect the function of the glycan shield. These data will advance our understanding of the molecular biology of HIV-1 and the factors that impact viral transmission and persistence. Ultimately, these studies will generate new information about Env vulnerabilities that can be exploited to develop future therapeutic approaches.