Conformational Landscape of the HIV-1 Envelope Glycoproteins

 性状（由申请方提供）：人免疫缺陷病毒（HIV-1）包膜糖蛋白（Envs）介导病毒进入宿主细胞。Env刺突是暴露在病毒表面上的唯一病毒特异性组分，因此代表了小分子进入抑制剂和疫苗诱导抗体的关键靶标。Env刺突是由三个gp 120外部Env和三个gp 41跨膜Env组成的三聚体。为了逃避宿主中和抗体，未配体的Env刺突呈现“闭合”构象（状态1）。在病毒进入过程中，储存在封闭Env构象中的高势能被引导到病毒和靶细胞膜的最终融合中。Env刺突通过与靶细胞上的第一受体CD 4结合而“打开”，导致形成前发夹中间体（状态3）。与第二受体CCR 5或CXCR 4结合，导致形成非常稳定的gp 41六螺旋束（状态4）以及病毒和宿主细胞膜的融合。 最近的证据表明存在多个“宽松的中间状态”（状态2a、2b等）。在未配体的闭合Env构象（状态1）和前发夹中间体（状态3）之间。HIV-1病毒株在进行转化的倾向上有所不同 从状态1到松弛的中间状态;更易分解或“反应性”的Env更可能对松弛的中间状态进行采样。“Env反应性”的这些差异影响HIV-1的嗜性和对小分子和抗体抑制的敏感性。因此，定义HIV-1 Env变异体的构象景观对于理解HIV-1生物学和指导干预措施的发展至关重要。 该提案的总体目标是了解对病毒进入至关重要的HIV-1 Env的构象转变。三个特定目的中的每一个都研究了HIV-1进入过程的不同阶段：1）特定目的1测试了Env三聚体上特定结构的修饰释放了维持构象状态1的限制的假设，允许Env对放松的中间状态（状态2a，2b等）进行采样。将确定Env变化对HIV-1对靶细胞受体水平的依赖性以及对小分子和抗体抑制的敏感性的影响。 2)特异性目的2使用依赖于还原剂的感染性的gp 41半胱氨酸取代突变体来检验gp 41卷曲螺旋在前发夹中间体（状态3）中以有序方式形成的假设。将鉴定由gp 41定向进入抑制剂靶向的Env的构象。3)具体目标3将定义HIV-1适应在表达非常低水平的CCR 5辅助受体的CD 4阳性细胞中复制的Env变化和机制。 上述研究将阐明HIV-1 Env膜融合机器的动态构象转变，并将加速HIV-1进入抑制剂的开发。

 DESCRIPTION (provided by applicant): The human immunodeficiency virus (HIV-1) envelope glycoproteins (Envs) mediate the entry of the virus into the host cell. The Env spike is the only virus-specific component exposed on the virus surface and thus represents a key target for small-molecule entry inhibitors and vaccine-induced antibodies. The Env spike is a trimer consisting of three gp120 exterior Envs and three gp41 transmembrane Envs. To evade host neutralizing antibodies, the unliganded Env spike assumes a "closed" conformation (State 1). During the virus entry process, the high potential energy stored in the closed Env conformation is channeled into the eventual fusion of the viral and target cell membranes. The Env spike is "opened" by binding to the first receptor on the target cell, CD4, resulting in the formation of a pre-hairpin intermediate (State 3). Binding to the second receptor, CCR5 or CXCR4, leads to the formation of a very stable gp41 six-helix bundle (State 4) and the fusion of viral and host cel membranes. Recent evidence has suggested the existence of multiple "relaxed intermediate states" (States 2a, 2b, etc.) between the unliganded closed Env conformation (State 1) and the pre-hairpin intermediate (State 3). HIV-1 strains differ in their propensity to make the transition from State 1 to the relaxed intermediate states; more triggerable or "reactive" Envs are more likely to sample the relaxed intermediate states. These differences in "Env reactivity" influence HIV-1 tropism and sensitivity to inhibition by small molecules and antibodies. Thus, defining the conformational landscape available to HIV-1 Env variants is essential for understanding HIV-1 biology and for guiding the development of interventions. The overall goal of this proposal is to understand the conformational transitions of the HIV-1 Envs that are critical to virus entry. Each of the three Specific Aims investigates a different stage of the HIV-1 entry process: 1) Specific Aim 1 tests the hypothesis that modification of specific structures on the Env trimer releases the restraints that maintain conformational State 1, allowing Env to sample the relaxed intermediate states (States 2a, 2b, etc.). The effects of the Env changes on HIV-1 dependence on target cell receptor levels and sensitivity to inhibition by small molecules and antibodies will be determined. 2) Specific Aim 2 uses gp41 cysteine-substitution mutants that are dependent upon a reducing agent for infectivity to test the hypothesis that the gp41 coiled coil in the pre-hairpin intermedite (State 3) forms in an ordered fashion. Conformations of Env targeted by gp41-directed entry inhibitors will be identified. 3) Specific Aim 3 will define the Env changes and mechanism underlying HIV-1 adaptation to replicate in CD4-positive cells expressing very low levels of the CCR5 coreceptor. The above studies will illuminate the dynamic conformational transitions of the HIV-1 Env membrane-fusing machine and will expedite the development of inhibitors of HIV-1 entry.