Watching conformational rearrangements in picornavirus replication proteins

项目摘要 一些最重要的新的和(重新)出现的病原体是正链RNA病毒， 包括冠状病毒和微小核糖核酸病毒，肠道病毒D68，肠道病毒A71，甚至脊髓灰质炎病毒。 这些病毒可以直接使用它们的RNA基因组来指导一个大的多蛋白的合成，这 然后必须被蛋白质分解成其组成部分，包括衣壳蛋白和酶 对基因组复制和封装很重要。病毒RNA基因组相当小，因此 这些病毒已经进化出从本质上扩展其功能蛋白质组的策略。例如, 小核糖核酸病毒3C蛋白是一种多功能蛋白，具有蛋白酶活性，结合RNA调控 对协调复制和翻译过程很重要的序列，并结合 在病毒“复制细胞器”中发现的磷脂脂，其作用是保护病毒免受宿主的侵袭。 细胞防御。所有这些活动都编码在其20 kDa的小结构中。另一项战略是 扩大功能蛋白含量是为了使蛋白分解前体具有不同于其 完全加工的对应物。例如，3C也被发现是3CD蛋白的一部分，但3CD 蛋白质具有不同的蛋白酶专一性，并具有不同的RNA和脂质结合亲和力。3CD 蛋白质也有3D结构域；3D蛋白质是依赖于RNA的RNA聚合酶，但3CD 不具有聚合酶活性。3CD本身也能上调肌醇磷脂的生成 并诱导膜的增殖，这是复制细胞器生物发生的重要事件。怎么了？ 3CD的不同和紧急功能还知之甚少；X射线晶体结构表明 这个3CD仅仅是3C和3D蛋白质通过一个小的柔性连接物连接在一起的复合体。 我们提出，结构动力学，即采样多个结构构象的能力，是 在了解病毒蛋白质功能方面缺失的成分。我们认为，3C在以下范围内波动 许多构象，为3C提供访问和协调其许多功能的能力，并且我们 提出3CD波动为不同的构象，为其提供可供选择的功能。这些 动态漂移可以通过与RNA、脂质和蛋白质结合的相互作用而进一步改变 合作伙伴来协调病毒蛋白的功能。我们将评估这些蛋白质的结构动力学 通过溶液状态核磁共振光谱，它提供了原子级的细节 蛋白质从皮秒到第二时间尺度的运动，并补充这些研究 突变研究，功能分析和基于细胞的方法，以更好地了解 病毒生命周期中的蛋白质结构动力学。完成的工作将提供新的机会 合理的抗病毒策略，例如，通过寻找与替代蛋白质结合的分子 构象和/或扰乱功能上重要的运动，如已经验证的3D。

Project Summary Some of the most important new and (re)emerging pathogens are positive-strand RNA viruses, including coronavirus and picornaviruses Enterovirus D68, Enterovirus A71 and even poliovirus. These viruses can directly use their RNA genome to guide the synthesis of a large polyprotein, which must then be proteolyzed into its component parts, including the capsid proteins and enzymes important for genome replication and encapsidation. Virus RNA genomes are rather small, and so these viruses have evolved strategies to essentially expand their functional proteomes. For example, the picornavirus 3C protein is a multi-functional protein that has protease activity, binds RNA control sequences important for coordinating replication and translation processes, and binds phosphoinositide lipids found in virus “replication organelles”, which act to protect the virus from host cell defenses. All of these activities are encoded within its small 20 kDa structure. Another strategy to expand functional protein content is for proteolytic precursors to have different functions than their fully processed counterparts. For example, 3C is also found as part of the 3CD protein, but the 3CD protein has different protease specificity, and different RNA and lipid binding affinities. The 3CD protein also has a 3D domain; the 3D protein is the RNA-dependent RNA polymerase but 3CD does not possess polymerase activity. By itself, 3CD also upregulates phosphoinositide lipid production and induces membrane proliferation, events important for replication organelle biogenesis. How the different and emergent functions of 3CD arise is poorly understood; X-ray crystal structures indicate that 3CD is merely a composite of the 3C and 3D proteins joined together by a small flexible linker. We propose that structural dynamics, that is, the ability to sample multiple structural conformations, is the missing ingredient in understanding virus protein function. We propose that 3C fluctuates among many conformations, providing 3C the ability to access and coordinate its many functions, and we propose that 3CD fluctuates into different conformations, providing it with alternative functions. These dynamic excursions can be further modified by interactions with RNA, lipids and protein binding partners to coordinate virus protein function. We will evaluate these protein structural dynamics through solution-state nuclear magnetic resonance spectroscopy, which provide atomic-level detail of protein motions from the picosecond to second timescales, and complement these studies with mutagenesis studies, functional assays and cell-based approaches to better understand the roles of protein structural dynamics in the virus life cycle. The completed work will provide new opportunities for rational anti-viral strategies, for example, by finding molecules that bind to alternative protein conformations and/or disrupt functionally-important motions, as already validated for 3D.