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: 10000945
• 负责人: Jae Hyun Cho
• 金额: $ 31.36万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10000945
• 关键词: Affinity; Antiviral Agents; Binding; Binding Sites; Biological Assay; Biophysics; Birds; Cell Nucleus; Cells; Competitive Binding; Complex; Data; Development; Disease; Epidemic; Future; Goals; Health; Human; Immune response; In Vitro; Infection; Influenza A virus; Integration Host Factors; Kinetics; Knowledge; Mediating; Mission; Molecular; Molecular Conformation; N-terminal; NMR Spectroscopy; Nonstructural Protein; Pathogenicity; Phosphotransferases; Play; Process; Proline; Proteins; Public Health; Recording of previous events; Research; Resolution; Roentgen Rays; Role; Signal Pathway; Signaling Protein; Site; Spanish flu; Structural Models; Structure; Testing; United States National Institutes of Health; Viral; Virulence; Virulence Factors; Virus; Virus Diseases; Virus Replication; Zoonoses; antiviral immunity; base; experimental study; flexibility; influenzavirus; inhibitor/antagonist; molecular dynamics; pandemic disease; pandemic influenza; prevent; programs; protein protein interaction; proto-oncogene protein c-crk; seasonal influenza; swine influenza; targeted agent; therapeutic development; therapeutic target

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 p85 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 p85 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 p85 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 p85 subunit of PI3K, reveal its functional role in PI3K activation, and identify hotspot NS1 residues that interact with both CrkII and p85. 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 p85 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 p85 相互作用以激活 PI3K 信号通路。我们的理由 研究表明，对 1918 NS1 与 CrkII 和 p85 相互作用的机制理解将有助于 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 p85 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 p85 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。