HIV neutralization and mechanisms of cellular entry

了解完整病毒上的三聚体Env的分子结构对于了解病毒-细胞相互作用的分子机制和设计有效的抗击艾滋病毒/艾滋病的免疫原都是重要的。在过去两年中，我们采取了几个重要步骤，以实现我们的目标，即了解三聚体包膜糖蛋白尖峰的结构以及艾滋病毒进入和中和的结构基础。我们的工作首次确定了未连接状态的HIV-1膜表面三聚体gp120的结构，以及与广中和抗体B12的复合体以及与CD4和17b抗体的三元复合体的结构。我们证明了CD4结合导致Env三聚体的重大重组，导致每个gp120单体的向外旋转和位移。Gp120三聚体封闭和开放状态的发现为解释和理解与病毒入侵相关的包膜糖蛋白的构象变化提供了新的范式。将这些研究扩展到一些SIV毒株，为三聚体Env在未连接状态以及在中和抗体和非中和抗体的复合体中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，大多数毒株都显示出处于三聚体关闭状态的包膜糖蛋白。然而，我们发现了一种SIV毒株，即使在没有结合的CD4的情况下，Env也以结构性开放的状态表达。开放的构象表明，这种菌株应该不再需要CD4才能进入，事实上，这正是我们现在能够证明的。非依赖于CD4的病毒经常在人体的免疫特权区域被发现，比如中枢神经系统。我们的发现为CD4非依赖性病毒进入的分子机制提供了一个结构上的解释，并进一步建立了冷冻电子断层扫描可以用来发现完整病毒上显示的不同的、功能相关的Env的四级结构。在研究三聚体Env刺激物结构的同时，我们还开始了系统性的工作，以分析HIV在巨噬细胞和树突状细胞等抗原提呈细胞中的分布。用常规的透射电子显微镜从感染细胞的薄片图像中很容易检测到含有HIV-1的内部间隔，但这些间隔的来源、连通性和三维分布仍然存在争议。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HIV-1感染的原代人巨噬细胞中病毒的三维分布。IA-SEM是最近发展起来的一种用于全细胞纳米级三维成像的方法。使用IA-SEM，我们显示在感染的巨噬细胞中存在一个广泛的含有HIV-1的管状室的网络，直径150-200 nm，从含有组装的HIV-1病毒粒子的泡状室延伸到细胞表面的长度可达5微米。将这些研究扩展到树突状细胞也是有益的。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连接处传递时，艾滋病毒感染的效率大大提高。利用离子磨损扫描电子显微镜、电子断层扫描和超分辨光学显微镜，我们分析了细胞-细胞接触的空间结构以及成熟树突状细胞和T细胞之间形成的病毒学突触上HIV病毒粒子的分布。我们证明了T细胞被来自成熟树突状细胞的片状膜延伸所包裹，导致形成病毒学突触的屏蔽区。在突触内，从CD4+T细胞发出的丝状突起使其与树突状细胞中隔离在表面可达间隔的3D网络中的HIV病毒粒子接触。病毒在树突状细胞和T细胞的膜表面都被检测到，但病毒粒子并不是在突触处被动释放的；相反，病毒的转移需要T细胞CD4受体的参与。T细胞与细胞外环境的相对隔离，艾滋病毒转移部位的埋葬，以及T细胞依赖受体启动病毒粒子转移，突出了细胞间艾滋病毒传播的新方面，并为限制艾滋病毒/艾滋病的传播提出了新的方法。

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.