Structural Studies of Protein Subunits

小核糖核酸病毒是最大的动物病毒家族之一，包括心脏病毒、脊髓灰质炎病毒、犀牛病毒和甲型肝炎病毒。本研究的目的是研究小核糖核酸病毒感染的三个早期阶段：与细胞受体的相互作用，受体介导的病毒粒子的构象变化，以及病毒通过一些非衣壳蛋白篡夺正常细胞功能。主流观点认为，其中一些病毒进化出了“峡谷”区，以保护受体结合区免受免疫监视，而宿主衍生的“口袋因子”在峡谷下结合，以调节受体的相互作用。我们最近的结果与这些前提不一致，并使我们提出了另一种假设：我们提出峡谷不是免疫压力的进化结果，而是为了促进衣壳动力学而存在的，“口袋因素”可能是一种人工制品。这笔拨款将通过解决以下问题直接测试这些相反的假设：1)免疫是否塑造了衣壳结构以及抗体如何阻止病毒进入？我们将继续对抗体/病毒复合物进行结构研究，因为它们为未来的疫苗开发和峡谷的真正功能提供了必要的细节。我们还将制作仅重链抗体文库，直接测试峡谷假说的各个方面，并提供一种寻找病毒“阿基里斯之踵”的新方法。2)衣壳动力学的结构细节是什么？“口袋因素”是否起作用？在我们最近的工作中，我们发现HRV14病毒衣壳是非常动态的。我们将使用分子生物学、质谱学和低温透射电镜来进一步研究这种衣壳“呼吸”的细节。这些研究不仅将阐明我们所认为的峡谷的真正功能，而且还将深入了解病毒如何将其基因组进入宿主细胞。3)非衣壳蛋白的结构是什么？它们如何篡夺细胞功能？小核糖核酸病毒在复制的早期阶段以及它们如何篡夺正常细胞功能方面有所不同。我们将确定其中一些蛋白质的结构，以更好地了解这一过程以及复制复合体是如何组装的。这些蛋白质也是抗病毒药物的天然靶点。这些研究有可能改变我们对病毒感染过程早期步骤的看法，并使我们能够开发适用于其他病毒系统的工具。

Picornaviruses are among the largest of animal virus families and include cardio-, polio-, rhino-, and hepatitis A viruses. The objective of this proposal is to examine three early stages of picornavirus infection: the interaction with the cellular receptor, the receptor-mediated conformational changes in the virion, and the viral usurping of normal cellular functions via a number of non-capsid proteins. The dominant dogma is that some of these viruses have evolved 'canyon' regions to protect the receptor-binding region from immune surveillance and that host-derived 'pocket factors' bind under the canyons to modulate receptor interactions. Our recent results are inconsistent with these premises and led us to propose an alternative hypothesis: We propose that the canyon is not the evolutionary consequence of immunological pressure but exists to facilitate capsid dynamics and 'pocket factors' are probably an artifact. This grant will directly test these opposing hypotheses by addressing the following questions: 1) Has immunity shaped the capsid structure and how is viral entry blocked by antibodies? We will continue structural studies on antibody/virus complexes since they offer details necessary for future vaccine development and the true function of the canyon. We will also make heavy-chain-only antibody libraries to directly test aspects of the canyon hypothesis and offer a novel way to find the 'Achilles heel' of a virus. 2) What are the structural details of the capsid dynamics and do 'pocket factors' play a role? In our recent work, we found that the HRV14 viral capsid is remarkably dynamic. We will use molecular biology, mass spectroscopy, and cryo-TEM to further examine the details of this capsid 'breathing'. These studies not only will elucidate what we believe to be the true function of the canyon, but will also yield insight as to how the virus gets its genome into the host cell. 3) What are the structures of the non-capsid proteins and how do they usurp cellular function? Picornaviruses differ in aspects of the early stages of replication and how they usurp normal cell functions. We will determine the structures of some of these proteins to better understand this process and how the replication complexes are assembled. These proteins are also natural targets for antiviral agents. These studies have the potential of changing the way we view the early steps in the viral infection process and will allow us to develop tools that are applicable to other viral systems.