Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer.
Receptor Activation of HIV-1 Env Leads to Asymmetric Exposure of the gp41 Trimer.
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HIV-1 ENV的受体激活导致GP41夹子的不对称暴露。
DOI：
10.1371/journal.ppat.1006098
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发表时间：
2016-12
期刊：
影响因子：
6.7
通讯作者：
Root MJ
中科院分区：
文献类型：
--
作者：
Khasnis MD;Halkidis K;Bhardwaj A;Root MJ
HIV-1糖蛋白Env的结构重排促进病毒通过膜融合进入。Env是一个对称的同源三聚体，每个原聚体由表面亚基gp 120和跨膜亚基gp 41组成。与gp 120结合的细胞CD 4和趋化因子受体协调gp 41的构象变化，首先是扩展的前发夹中间体（PHI），最终是融合的发夹三聚体（TOH）。HIV-1融合抑制剂靶向PHI中的gp 41并阻断TOH形成。为了表征进入和通过PHI的结构转化，我们采用了含有针对个体融合抑制剂的高亲和力和低亲和力结合位点的不对称Env三聚体。使用由CD 4或趋化因子受体结合缺陷的原聚体组成的工程化Env异源三聚体实现不对称性。将受体结合与抑制剂亲和力联系起来，使我们能够在功能性三聚体的背景下评估单个Env原聚体的构象变化。我们发现，过渡到PHI可以发生对称或不对称的依赖于化学计量的CD 4结合。多个CD 4的顺序接合以CD 4依赖性方式促进单个融合抑制剂结合位点的渐进性暴露。相比之下，只有一个单一的CD 4分子的参与导致了延迟，但对称，暴露的gp 41三聚体。Env-CD 4相互作用和gp 41暴露之间的这种复杂耦合解释了对于具有天然不对称gp 41的突变Env同源三聚体观察到的多相融合抑制剂滴定。我们的研究结果表明，gp 41的空间和时间暴露可以进行在一个不一致的，不对称的方式取决于参与Env三聚体的CD 4的数量。这些发现对病毒膜融合的机制和开发旨在引发靶向PHI中gp 41的中和抗体的候选疫苗具有重要意义。对于艾滋病毒来说，细胞入侵需要合并病毒和细胞膜，这是通过病毒融合蛋白Env的活性实现的事件。Env由三个gp 120和三个gp 41亚基对称排列在病毒表面组成。gp 120亚基结合细胞受体，进而协调促进膜融合的gp 41构象变化。了解这些结构重排阐明了病毒膜融合的机制，也刺激了病毒进入的靶向抑制剂和引发抗病毒免疫应答的候选疫苗的开发。在这项研究中，我们采用了一种新的策略来研究功能性Env复合物中的单个亚基。该策略将不同的gp 120-受体相互作用与暴露特定gp 41亚基的构象变化联系起来。我们发现，尽管其亚基的初始对称排列，Env构象变化最经常进行相当不对称，导致暴露的gp 41三聚体的大部分融合事件。这一发现可能解释了为什么试图诱导有效的抗HIV抗体完全暴露的gp 41三聚体在很大程度上是不成功的。这项研究使我们得以一窥导致Env介导的膜融合的早期结构转变，并为询问其他膜封装病毒的融合蛋白提供了一个框架。
Structural rearrangements of HIV-1 glycoprotein Env promote viral entry through membrane fusion. Env is a symmetric homotrimer with each protomer composed of surface subunit gp120 and transmembrane subunit gp41. Cellular CD4- and chemokine receptor-binding to gp120 coordinate conformational changes in gp41, first to an extended prehairpin intermediate (PHI) and, ultimately, into a fusogenic trimer-of-hairpins (TOH). HIV-1 fusion inhibitors target gp41 in the PHI and block TOH formation. To characterize structural transformations into and through the PHI, we employed asymmetric Env trimers containing both high and low affinity binding sites for individual fusion inhibitors. Asymmetry was achieved using engineered Env heterotrimers composed of protomers deficient in either CD4- or chemokine receptor-binding. Linking receptor engagement to inhibitor affinity allowed us to assess conformational changes of individual Env protomers in the context of a functioning trimer. We found that the transition into the PHI could occur symmetrically or asymmetrically depending on the stoichiometry of CD4 binding. Sequential engagement of multiple CD4s promoted progressive exposure of individual fusion inhibitor binding sites in a CD4-dependent fashion. By contrast, engagement of only a single CD4 molecule led to a delayed, but symmetric, exposure of the gp41 trimer. This complex coupling between Env-CD4 interaction and gp41 exposure explained the multiphasic fusion-inhibitor titration observed for a mutant Env homotrimer with a naturally asymmetric gp41. Our results suggest that the spatial and temporal exposure of gp41 can proceed in a nonconcerted, asymmetric manner depending on the number of CD4s that engage the Env trimer. The findings have important implications for the mechanism of viral membrane fusion and the development of vaccine candidates designed to elicit neutralizing antibodies targeting gp41 in the PHI. For HIV, cellular invasion requires merging viral and cellular membranes, an event achieved through the activity of the viral fusion protein Env. Env consists of three gp120 and three gp41 subunits symmetrically arranged on the viral surface. The gp120 subunits bind cellular receptors, which, in turn, coordinate gp41 conformational changes that promote membrane fusion. Understanding these structural rearrangements illuminates the mechanism of viral membrane fusion, and also spurs development of targeted inhibitors of viral entry and vaccine candidates that elicit antiviral immune responses. In this study, we employed a novel strategy to investigate individual subunits in the context of functioning Env complexes. The strategy links distinct gp120-receptor interactions to conformational changes that expose specific gp41 subunits. We found that, despite the initial symmetric arrangement of its subunits, Env conformational changes most often proceed quite asymmetrically, leading to exposure of only one-third of the gp41 trimer for much of the fusion event. This finding might explain why attempts to elicit potent anti-HIV antibodies to a fully exposed gp41 trimer have been largely unsuccessful. The study gives us a glimpse of the early structural transitions leading to Env-mediated membrane fusion and provides a framework for interrogating the fusion proteins of other membrane-encapsulated viruses.