Role of signal sequence variation in governing HIV Env Functions

项目概要： HIV-1与宿主细胞的初始相互作用由病毒包膜（Env）刺突介导， 其由三个GP 120-GP 41异二聚体组成。由于病毒Env是存在于细胞表面的唯一病毒抗原， 病毒表面，它是宿主抗体（Ab）反应的唯一靶标。HIV-1 Env的一个标志是其 重糖基化。事实上，聚糖占gp 120亚基分子量的一半。n-连接 聚糖对于Env的正确折叠是必需的，并且围绕每个gp 120的>25个N-聚糖 已知亚基屏蔽Ab表位。然而，人们对这些机制和因素知之甚少， 影响聚糖占据率和修饰病毒的聚糖类型（高甘露糖、杂合、复合） Env.此外，N-连接的聚糖，特别是它们的糖组成在调节糖代谢中的重要性， 该病毒的传染性和传播性尚未完全了解。 在该提议中，我们专注于研究Env信号序列（SS）变化在 调节HIV Env功能和糖基化。党卫队已经被证明在 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Env可变环一样高度可变，并且在不同的SS之间观察到显著的序列差异。 T/F vs.急性vs.慢性病毒，以及Ab敏感1级病毒和Ab耐药2级病毒的SS之间， 三级病毒。我们的初步数据表明，Env-SS中的单个氨基酸变化足以 通过靶向HIV-1包膜上经常被掩蔽的表位的单克隆抗体， V2和V3循环。Env-SS突变还通过DC-SIGN影响病毒的反式感染，DC-SIGN结合N- HIV-1 Env.因此，我们提出了一个总体假设，即Env SS是一种活性的 HIV-1 Env功能的调节剂。Env SS中的突变影响病毒感染性，通过DC- SIGN和穿过上皮屏障，以及通过调节N- 病毒Env的聚糖占有率和糖组成。为了验证这一假设，我们将确定 T/F 2级病毒与其他病毒（T/F、慢性、1级、2级）中发现的HIV Env SS多态性的影响 在调节病毒感染性，传播，抗原性，中和使用细胞为基础的， 免疫化学测定（Aim 1）。我们将进一步评估Env SS变化是否与 使用寡糖特异性凝集素和高能C-陷阱解离改变N-聚糖组成 质谱法（HCD-MS）（目的2）。这些研究将使我们更好地了解 SS多态性在影响HIV-宿主相互作用中的作用，并为设计 开发更有效的HIV疫苗Env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.