HIV neutralization and mechanisms of cellular entry

了解完整病毒上三聚体Env的分子结构并描述传播机制对于设计有效的免疫原和治疗剂以对抗HIV/AIDS至关重要。此外，相关的包膜病毒，如流感病毒和埃博拉病毒可能具有相似的病毒进入机制，因此这些病毒的结构研究可能为所有这三种病毒的疫苗设计提供见解。去年，我们继续在实现这些目标方面取得重大进展。HIV-1感染始于三聚体病毒包膜糖蛋白（Env）与CD 4和靶T细胞上的辅助受体结合。了解这些配体如何影响Env的结构对于HIV疫苗开发具有根本意义。使用冷冻电子显微镜，我们已经确定了大量的天然HIV-1 Env在其未结合状态，结合可溶性CD 4，或结合中和抗体的结构。这些研究描述了一系列的构象之前的前发夹状态的复合物。使用切割的溶解形式的HIV-1 Env，我们能够在其融合前（6埃分辨率）和开放的活化（9埃分辨率）构象中实现三聚体的更高分辨率结构，揭示了在刺突开放期间外部gp 120亚基围绕三个内部螺旋旋转。我们正在继续探索各种不同中和抗体的中和机制，并确定结合的哪些方面导致中和潜力。在过去的一年中，我们已经将这些相同的技术应用于埃博拉包膜糖蛋白和流感HA三聚体，因为这些包膜病毒在结构上具有相似性，并且可能具有相似的进入机制。埃博拉病毒是一种新兴病原体，已成为疫苗和治疗开发的关键目标。埃博拉病毒在成熟病毒体的表面上展示了单一复合物（包膜糖蛋白）的许多拷贝。针对包膜糖蛋白的广泛中和抗体已被证明在预防病毒融合方面有效;然而，包膜糖蛋白的某些结构特征，如粘蛋白结构域，似乎增强了埃博拉病毒的感染性，仍然知之甚少。理解粘蛋白结构域的位置是至关重要的，特别是考虑到许多中和抗体与该区域紧密结合。使用冷冻电子断层扫描，我们确定了埃博拉糖蛋白的第一个结构，显示粘蛋白结构域，一个大的和高度异质性的区域，可以阻碍抗体结合。在这项工作的基础上，我们现在正在努力确定埃博拉病毒包膜糖蛋白的结构，这些糖蛋白与目前用作病毒治疗剂的抗体结合。这些结构可以提供对治疗和/或预防这种病毒感染的最有效机制的见解。两年前，我们报道了使用冷冻电子断层扫描首次确定与中和抗体C179结合的2009年大流行毒株上的天然流感HA三聚体的结构。在这些研究的继续中，我们正在探索HA三聚体的不同变体之间的结构差异，目的是发现在通用流感疫苗中最能引发中和抗体的结构元件。为了进一步了解HIV-1感染过程，我们还对HIV-1核心生物发生进行了结构研究。携带病毒遗传物质的HIV-1核心与HIV-1病毒粒子同时形成。在核心形成的初始阶段，HIV-1 gag多蛋白在出芽病毒体的内表面上聚结，形成晶格结构。一系列的裂解事件，这是必要的适当的核心形成，释放衣壳亚基，允许成熟的核心形成。核心形成的经典模型表明，这些亚基在自发成核之前作为单体释放到形成病毒体的内腔中，从窄端生长成有序结构。我们使用冷冻电子断层扫描可视化核心内的病毒粒子以及多核心含有来自HIV-1细胞培养上清液的隔室。这些断层图像揭示了一些关键特征：首先，原生岩心不显示经典模型所建议的完全有序的结构。相反，核心有许多缺陷，以及几何形状不太可能形成的成核和生长模型。更重要的是，我们的成像还显示了似乎部分形成的结构，滚动的核心，拴在膜的内部。这些发现使我们提出了一个新的核心形成模型：我们提出，在裂解的衣壳蛋白从gag晶格，释放应力允许衣壳亚基进行非扩散相变的成熟的晶格结构。这种转变反过来又导致衣壳片卷曲远离膜，卷成经典的圆锥形核心形状，同时由于相变的失配而留下许多裂缝和其他缺陷。

Knowledge of the molecular structure of trimeric Env on intact viruses and delineating the mechanisms of transmission are central to the design of effective immunogens and therapeutic agents to combat HIV/AIDS. In addition, related enveloped viruses such as influenza and Ebola may share similar mechanisms for viral entry, and as such structural studies of these viruses may offer insight towards vaccine design for all three of these viruses. We have continued to make significant progress towards these goals over the last year. HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target T-cells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we have determined a large number of structures for native HIV-1 Env in its unbound state, bound to soluble CD4, or bound to neutralizing antibodies. These studies described a series of conformations prior to the pre-hairpin state of the complex. Using a cleaved, solubilized version of the HIV-1 Env, we were able to achieve higher resolution structures of the trimer in its pre-fusion (6 Angstrom resolution) and open, activated (9 Angstrom resolution) conformations, revealing that the outer gp120 subunits rotate around three internal helices during spike opening. We are continuing to explore the mechanism for neutralization by a variety of different neutralizing antibodies, and to determine what aspects of binding lead to neutralizing potential. In the past year, we have applied these same techniques to Ebola envelope glycoprotein and to influenza HA trimers, as these enveloped viruses have similarities in structure, and may share a similar mechanism for entry. The Ebola virus is an emerging pathogen that has become a critical target for vaccine and therapeutic development. Ebola displays many copies of a single complex, the envelope glycoprotein, on the surface of mature virions. Broadly neutralizing antibodies directed at the envelope glycoprotein have proven effective in preventing viral fusion; however, certain structural features of the envelope glycoprotein, such as the mucin domains, which appear to enhance Ebola infectivity, have remained poorly understood. Understanding the placement of the mucin domain is critical, especially considering that many neutralizing antibodies bind in close proximity to this region. Using cryo-electron tomography, we determined the first structure of the Ebola glycoprotein showing the mucin domain, a large and highly heterogeneous region that can impede antibody binding. Building upon this work, we are now working to determine the structure of Ebola envelope glycoprotein bound to antibodies currently being used as therapeutics for the virus. These structures may offer insight into the most effective mechanism to treating and/or preventing infection by this virus. Two years ago we reported the first determination of the structure of the native influenza HA trimer on the 2009 pandemic strain bound to the neutralizing antibody C179 using cryo-electron tomography. In continuation of these studies, we are exploring differences in structure between different variants of HA trimers with the goal of discovering the structural elements that will best elicit neutralizing antibodies in a universal influenza vaccine. To further understand the HIV-1 infection process, we have also undertaken a structural study of the HIV-1 core biogenesis. The HIV-1 core, which carries the viral genetic material, forms at the same time as the HIV-1 virion. In the initial stages of core formation, the HIV-1 gag polyprotein coalesces on the internal surface of a budding virion, forming a lattice structure. A series of cleavage events, which are required for proper core formation, release the capsid subunits, allowing the mature core to form. Classical models for core formation have suggested that these subunits release as monomers into the lumen of the forming virion, before spontaneously nucleating, growing into an ordered structure from the narrow end. We used cryo-electron tomography to visualize cores within virions as well as multi-core containing compartments derived from HIV-1 cell culture supernatants. These tomograms revealed a number of key features: first, native cores do not display the entirely ordered structure suggested by the classical models. Instead, the cores have numerous defects, as well as geometries unlikely to form by nucleation and growth models. More importantly, our imaging also revealed structures that appear to be partially formed, rolling cores, tethered to the inside of the membrane. These findings led us to suggest a new model for core formation: we propose that during cleavage of the capsid protein from the gag lattice, the release of stresses allows the capsid subunits to undergo a non-diffusional phase transition to the mature lattice structure. This transition in turn causes the capsid sheet to curl away from the membrane, rolling into the classical conical core shape, while leaving numerous cracks and other defects due to mismatches from the phase transition.