Role of signal sequence variation in governing HIV Env Functions

项目总结： HIV-1与宿主细胞的初始相互作用是由病毒包膜(Env)尖峰介导的，每个 它由三个gp120-gp41杂二聚体组成。因为病毒包膜是唯一存在于 病毒表面，它是宿主抗体(Ab)反应的唯一靶点。HIV-1环境的一个标志是它的 严重的糖基化作用。事实上，多聚糖构成了gp120亚基分子质量的一半。N-连锁的 多糖对于环境的正确折叠是必不可少的，而25个N-糖链包裹着每个gp120 亚基是已知的屏蔽抗体表位的单位。然而，人们对这一机制和因素知之甚少。 影响糖链占有率和修饰病毒的糖链类型(高甘露糖、杂交链、复合链) 环境此外，N-连接的多聚糖，特别是它们的糖组成在调节 病毒的传染性和传播性尚不完全清楚。 在这项建议中，我们重点研究环境信号序列(SS)变异在 调节HIV Env的功能和糖基化。党卫军已被证明在 糖蛋白的生物合成、糖基化和构象折叠。值得注意的是，HIV Env SS是 与环境变量循环一样高度可变，并且观察到在 T/F与急性病毒与慢性病毒的比较，以及抗体敏感的第一级病毒的SS与抗抗体的第二级病毒和 第三级病毒。我们的初步数据显示，Env-SS中的单个氨基酸变化足以 针对通常被屏蔽的表位的单抗显著改变HIV-1环境识别和病毒中和 V2和V3循环。Env-SS突变还通过与N-S结合的DC-SIGN影响病毒的反式感染。 HIV-1包膜上的葡聚糖因此，我们提出了一个总体假设，即环境SS是一个主动的 HIV-1env功能的调节剂。Env SS中的突变会影响病毒的传染性，通过DC- 信号和跨上皮屏障，通过调节N-受体识别和中和抗体 病毒环境中的糖链占有率和糖组成。为了检验这一假设，我们将确定 在T/F第二级病毒中发现的HIV Env SS多态性与其他病毒(T/F、慢性、第一级、第二级)的影响 在调节病毒的传染性、传播性、抗原性和中和性方面，使用基于细胞的和 免疫化学分析(目标1)。我们将进一步评估环境SS变异是否与 利用寡糖特异性凝集素和高能C-陷阱解离改变N-糖链的组成 质谱仪(HCD-MS)(目标2)。这些研究将使我们更好地理解 SS基因多态性在影响HIV-宿主相互作用中的作用并为设计提供有价值的数据 为HIV疫苗开发更有效的环境免疫原的新策略。

Project summary: The initial interaction of HIV-1 with the host cells is mediated by the virus envelope (Env) spike, each of which is composed of three gp120-gp41 heterodimers. Since the virus Env is the only viral antigen present on the virus surface, it is the sole target for the host antibody (Ab) responses. One hallmark of the HIV-1 Env is its heavy glycosylation. Indeed, glycans comprise half of the molecular mass of the gp120 subunit. The N-linked glycans are essential for the proper folding of the Env, and the >25 N-glycans shrouding each of the gp120 subunit are known to shield Ab epitopes. However, very little is known about the mechanisms and factors that influence the glycan occupancy and the types of glycans (high-mannose, hybrid, complex) decorating the virus Env. Moreover, the importance of N-linked glycans and especially their sugar compositions in regulating the infectivity and transmissibility of the virus is not fully understood. In this proposal we focus on studying the contribution of the Env signal sequence (SS) variations in modulating HIV Env functions and glycosylation. The SS has been shown to play a crucial role in the biosynthesis, glycosylation and conformational folding of glycoproteins in general. Notably, the HIV Env SS is as highly variable as the Env variable loops, and prominent sequence differences are observed between SS of T/F vs. acute vs. chronic viruses, and between SS of Ab-sensitive Tier 1 viruses and Ab-resistant Tier 2 and Tier 3 viruses. Our preliminary data show that a single amino-acid change in the Env-SS is sufficient to drastically alter HIV-1 Env recognition and virus neutralization by MAbs that target often masked epitopes on the V2 and V3 loops. The Env-SS mutations also affect virus trans-infection via DC-SIGN, which binds to N- glycans on the HIV-1 Env. Hence, we propose an overall hypothesis that the Env SS is an active modulator of HIV-1 Env functions. Mutations in the Env SS affect virus infectivity, transmission via DC- SIGN and across epithelial barrier, and antibody recognition and neutralization by modulating the N- glycan occupancy and sugar compositions of the virus Env. To test this hypothesis, we will determine the impact of HIV Env SS polymorphisms found in a T/F Tier 2 virus vs. other viruses (T/F, chronic, Tier 1, Tier 2) in modulating the virus infectivity, transmission, antigenicity, and neutralization using cell-based and immunochemical assays (Aim 1). We will further evaluate whether Env SS variations are associated with alterations of N-glycan compositions using oligosaccharide-specific lectins and high energy C-trap dissociation mass spectrometry (HCD-MS) (Aim 2). These studies will lead us to a better understanding about the contribution of SS polymorphisms in influencing HIV-host interactions and provide valuable data for designing novel strategies to develop more effective Env immunogens for HIV vaccines.