Structure and functional dynamics of virus-host protein interactions

病毒-宿​​主蛋白相互作用的结构和功能动力学

• 批准号: 10581354
• 负责人: Jae Hyun Cho
• 金额: $ 15.28万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-20 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581354
• 关键词: Affinity; Antiviral Agents; Binding; Biological Assay; Biophysics; Birds; Cell Nucleus; Cells; Complex; Disease; Future; Goals; Health; Human; Immune response; Influenza A virus; Knowledge; Mediating; Mission; Molecular; NMR Spectroscopy; Nonstructural Protein; Pathogenicity; Play; Process; Proline; Proteins; Public Health; Recording of previous events; Research; Roentgen Rays; Role; Signal Pathway; Signaling Protein; Site; Spanish flu; Structure; Testing; United States National Institutes of Health; Viral; Virulence Factors; Virus; Virus Replication; Zoonoses; antiviral drug development; base; experimental study; influenzavirus; inhibitor; molecular dynamics; pandemic influenza; programs; swine influenza; targeted agent; therapeutic development

The 1918 influenza A virus (IAV), also known as the Spanish flu, caused the worst influenza pandemic in human history. Nonstructural protein 1 (NS1) is a multifunctional virulence factor associated with the suppression of anti- viral immune responses and thereby has been identified as one of the molecular determinants of high pathogenicity of the 1918 IAV. NS1 of the 1918 IAV (1918 NS1) contains a proline-rich motif (PRM) that mediates binding with host CrkII with high affinity and selectivity. The 1918 NS1:CrkII interaction plays critical roles in the suppression of host anti-viral immune responses and the enhancement of viral replication. Moreover, NS1s of many avian/swine IAVs contain the CrkII-binding PRM. Given the zoonotic potential of IAVs, there is a critical need to determine the molecular mechanisms by which the interaction of 1918 NS1 and cellular CrkII is regulated. The long-term goal of our research program is to elucidate the molecular mechanisms underlying virus-host protein interactions. Our objectives in this proposal are to determine the structural mechanisms of the 1918 NS1:CrkII interaction, and to determine the molecular and cellular mechanisms whereby the 1918 NS1:CrkII complex induces PI3K activation, resulting in enhanced viral replication. Our central hypothesis is that the 1918 NS1:CrkII complex is structurally dynamic, which is functionally important for the interaction with the p85β regulatory subunit of PI3K. To test this hypothesis, we will determine the structure of the 1918 NS1:CrkII complex and elucidate how the complex interacts with p85β to activate the PI3K signaling pathway. Our rationale for these studies is that the mechanistic understanding of the interactions of 1918 NS1 with CrkII and p85β would help identify previously undiscovered target sites to develop for potential inhibitors against the 1918 NS1. Through a synergistic approach combining small-angle X-ray scattering, NMR spectroscopy, molecular dynamics simulation, and cell-based assays, we will pursue the following specific aims. Aim 1. To determine the structural mechanism of the 1918 NS1:CrkII interaction using a battery of biophysical experiments. Hijacking and relocation of CrkII into the nucleus is a distinctive feature of the 1918 pandemic IAV NS1. To understand this process, we will reveal structural and energetic mechanisms by which the affinity and lifetime (1/koff) of the 1918 NS1:CrkII complex are modulated. Aim 2. To determine the molecular mechanism underlying NS1-induced PI3K activation. The 1918 NS1:CrkII interaction markedly enhances NS1-induced PI3K activation; however, its molecular mechanism is unknown. We will seek to comprehensively determine the molecular mechanisms by which the 1918 NS1:CrkII complex interacts with the p85β subunit of PI3K, reveal its functional role in PI3K activation, and identify hotspot NS1 residues that interact with both CrkII and p85β. This study is expected to have a positive impact on the development of anti-viral agents targeting NS1-host protein interactions.

1918年甲型流感病毒（IAV），也称为西班牙流感，引起了人类最严重的流感大流行 历史。非结构蛋白 1 (NS1) 是一种多功能毒力因子，与抑制抗- 病毒免疫反应，因此已被确定为高病毒免疫反应的分子决定因素之一 1918 年 IAV 的致病性。 1918 IAV (1918 NS1) 的 NS1 包含富含脯氨酸的基序 (PRM)，可介导 与宿主 CrkII 具有高亲和力和选择性结合。 1918 NS1:CrkII 相互作用在 抑制宿主抗病毒免疫反应并增强病毒复制。此外，NS1 许多禽/猪 IAV 含有 CrkII 结合 PRM。鉴于 IAV 的人畜共患潜力，存在一个关键问题 第1918章 被人欺负了 我们研究计划的长期目标是阐明病毒宿主的分子机制 蛋白质相互作用。我们本提案的目标是确定 1918 年的结构机制 NS1:CrkII 相互作用，并确定 1918 NS1:CrkII 的分子和细胞机制 复合物诱导 PI3K 激活，从而增强病毒复制。我们的中心假设是 1918 年 NS1:CrkII 复合物在结构上是动态的，这对于与 p85β 的相互作用具有重要的功能 PI3K 的调节亚基。为了验证这一假设，我们将确定 1918 NS1:CrkII 复合物的结构 并阐明该复合物如何与 p85β 相互作用以激活 PI3K 信号通路。我们的理由 研究表明，了解 1918 NS1 与 CrkII 和 p85β 相互作用的机制将有助于 确定以前未发现的靶位点，以开发针对 1918 NS1 的潜在抑制剂。通过一个 结合小角 X 射线散射、核磁共振波谱、分子动力学的协同方法 模拟和基于细胞的测定，我们将追求以下具体目标。目标 1. 确定结构 使用一系列生物物理实验研究了 1918 NS1:CrkII 相互作用的机制。劫持和搬迁 CrkII 进入细胞核是 1918 年大流行 IAV NS1 的一个显着特征。为了理解这个过程，我们 将揭示 1918 NS1:CrkII 的亲和力和寿命 (1/koff) 的结构和能量机制 复杂的调制。目标 2. 确定 NS1 诱导 PI3K 激活的分子机制。 1918 NS1:CrkII 相互作用显着增强 NS1 诱导的 PI3K 激活；然而，它的分子 机制未知。我们将寻求全面确定其分子机制 1918 NS1:CrkII 复合物与 PI3K 的 p85β 亚基相互作用，揭示其在 PI3K 激活中的功能作用，并 识别与 CrkII 和 p85β 相互作用的热点 NS1 残基。这项研究预计将产生积极作用 对针对 NS1-宿主蛋白相互作用的抗病毒药物的开发产生影响。