HIV neutralization and mechanisms of cellular entry

了解完整病毒上三聚体Env的分子结构对于理解病毒-细胞相互作用的分子机制以及设计有效的免疫原来对抗HIV/AIDS非常重要。在过去的两年中，我们朝着了解三聚包膜糖蛋白尖峰结构和HIV进入和中和的结构基础的目标迈出了几项重要的步骤。我们的工作首次确定了HIV-1膜表面的三聚体gp120在非配体状态下的结构，与广泛中和的抗体b12复合物以及与CD4和17b抗体的三元复合物。我们证明CD4结合导致Env三聚体的主要重组，导致每个gp120单体向外旋转和位移。gp120三聚体的封闭和开放状态的发现为解释和理解与病毒进入相关的包膜糖蛋白的构象变化提供了新的范式。将这些研究扩展到许多SIV菌株，为三聚体Env在非配体状态和与中和和非中和抗体复合物的分子组织提供了新的和意想不到的见解。对十几种不同的HIV和SIV毒株的比较表明，大多数显示三聚体处于封闭状态的包膜糖蛋白。然而，我们已经发现一种SIV毒株，即使在没有结合CD4的情况下，Env也以构成性开放状态表达。开放的构象表明，这种菌株应该不再需要CD4进入，事实上，这正是我们现在能够证明的。不依赖cd4的病毒通常存在于人体的免疫特权区域，如中枢神经系统。我们的发现为不依赖cd4的病毒进入的分子机制提供了结构上的解释，并进一步确立了冷冻电子断层扫描可以用来发现完整病毒上显示的Env的不同的、功能相关的四级结构。在研究三聚体Env刺突结构的同时，我们也开始系统地分析HIV在抗原呈递细胞（如巨噬细胞和树突状细胞）中的分布。利用传统的透射电子显微镜可以很容易地在感染细胞的薄片图像中检测到含有hiv -1的内部区室，但是这些区室的起源、连通性和3D分布仍然存在争议。我们最近使用冷冻电子断层扫描和离子磨损扫描电子显微镜（IA-SEM）确定了病毒在hiv -1感染的原代人巨噬细胞中的3D分布，这是一种最近开发的全细胞纳米级3D成像方法。利用IA-SEM，我们发现在被感染的巨噬细胞中存在一个广泛的含有HIV-1的管状腔室网络，直径为150- 200nm，长度可达5nm，从含有组装HIV-1病毒粒子的囊泡腔室延伸到细胞表面。将这些研究扩展到树突状细胞也提供了信息。当病毒通过称为病毒学突触的特殊结构在CD4+ t细胞和携带病毒的树突状细胞之间的偶联物传递时，HIV感染的效率大大提高。利用离子磨损扫描电镜、电子断层扫描和超分辨率光学显微镜，我们分析了细胞-细胞接触的空间结构和成熟树突状细胞和t细胞之间形成的病毒学突触上HIV病毒粒子的分布。我们展示了来自成熟树突状细胞的片状膜延伸对t细胞的惊人包膜，从而形成了一个屏蔽区域，用于形成病毒学突触。在突触内，从CD4+ t细胞发出的丝状延伸与树突状细胞表面可接近的三维空间网络深处的HIV病毒接触。树突状细胞和t细胞的膜表面都能检测到病毒，但病毒粒子不会在突触被动释放；相反，病毒转移需要t细胞CD4受体的参与。t细胞与细胞外环境的相对隔离、HIV转移位点的埋藏以及t细胞依赖受体启动病毒粒子转移突出了细胞-细胞HIV传播的新方面，并提出了限制HIV/AIDS传播的新方法。

Knowledge of the molecular structure of trimeric Env on intact viruses is important both for understanding the molecular mechanisms underlying virus-cell interactions and for the design of effective immunogens to combat HIV/AIDS. We have taken several important steps over the last two years towards our goal of understanding the structure of trimeric envelope glycoproteins spikes and the structural basis of HIV entry and neutralization. Our work has resulted in the first determination of the structure of trimeric gp120 on the surface of the HIV-1 membrane in the unliganded state, in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. We demonstrated that CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This discovery of the closed and open states of the gp120 trimer has provided a new paradigm to interpret and understand the conformational changes of envelope glycoproteins relevant to viral entry. Extension of these studies to a number of SIV strains is providing new and unexpected insights into the molecular organization of trimeric Env in unliganded states and in complex with both neutralizing and non-neutralizing antibodies. Comparison of over a dozen different HIV and SIV strains shows that most display envelope glycoproteins that are in the closed state of the trimer. However, we have discovered an SIV strain where Env is expressed in a constitutively open state even in the absence of bound CD4. The open conformation suggests that this strain should no longer require CD4 for entry, and indeed this is exactly what we have now been able to demonstrate. CD4-independent viruses are often found in immune-privileged areas of the body such as the central nervous system. Our findings suggest a structural explanation for the molecular mechanism of CD4-independent viral entry and further establish that cryo-electron tomography can be used to discover distinct, functionally relevant quaternary structures of Env displayed on intact viruses. In parallel with structural investigation of trimeric Env spikes, we have also initiated systematic efforts to analyze HIV distribution in antigen presenting cells such as macrophages and dendritic cells. HIV-1-containing internal compartments are readily detected in images of thin sections from infected cells using conventional transmission electron microscopy, but the origin, connectivity and 3D distribution of these compartments has remained controversial. We recently determined the 3D distribution of viruses in HIV-1-infected primary human macrophages using cryo-electron tomography and ion-abrasion scanning electron microscopy (IA-SEM), a recently developed approach for nanoscale 3D imaging of whole cells. Using IA-SEM we showed the presence of an extensive network of HIV-1-containing tubular compartments in infected macrophages, with diameters of 150-200 nm, and lengths of up to 5 mum that extend from vesicular compartments that contain assembling HIV-1 virions to the cell surface. Extension of these studies to dendritic cells has also been informative. The efficiency of HIV infection is greatly enhanced when the virus is delivered at conjugates between CD4+ T-cells and virus-bearing dendritic cells via specialized structures known as virological synapses. Using ion abrasion scanning electron microscopy, electron tomography, and super-resolution light microscopy, we have analyzed the spatial architecture of cell-cell contacts and distribution of HIV virions at virological synapses formed between mature dendritic cells and T-cells. We demonstrate the striking envelopment of T-cells by sheet-like membrane extensions derived from mature dendritic cells, resulting in a shielded region for formation of virological synapses. Within the synapse, filopodial extensions emanating from CD4+ T-cells make contact with HIV virions sequestered deep within a 3D network of surface-accessible compartments in the dendritic cell. Viruses are detected at the membrane surfaces of both dendritic cells and T-cells, but virions are not released passively at the synapse; instead virus transfer requires the engagement of T-cell CD4 receptors. The relative seclusion of T-cells from the extracellular milieu, the burial of the site of HIV transfer and the receptor-dependent initiation of virion transfer by T-cells highlight novel aspects of cell-cell HIV transmission, and suggest new approaches for limiting the spread of HIV/AIDS.