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: 10473691
• 负责人: Jae Hyun Cho
• 金额: $ 30.77万
• 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/10473691
• 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 drug development; antiviral immunity; base; experimental study; flexibility; influenzavirus; inhibitor; 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年的影响力（IAV），也称为西班牙流感，引起了人类最严重的影响。 历史。非结构蛋白1（NS1）是与抑制抗抑制有关的多功能病毒因子 病毒免疫反应，因此被确定为高的分子决定剂之一 1918 IAV的致病性。 1918年IAV（1918 NS1）的NS1包含一个富含脯氨酸的基序（PRM） 与具有高亲和力和选择性的宿主CRKII结合。 1918年NS1：CRKII相互作用在 抑制宿主抗病毒免疫反应和病毒复制的增强。而且，NS1S 许多鸟/猪IAV都包含CRKII结合PRM。鉴于IAV的人畜共患潜力，有一个关键 需要确定调节1918 NS1和细胞CRKII相互作用的分子机制。 我们的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。我们在此提案中的目标是确定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的相互作用在功能上很重要 PI3K的监管亚基。为了检验这一假设，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。我们对这些的理由 研究是，对1918 NS1与CRKII和P85的相互作用的机械理解将有助于 确定先前未发现的目标位点，以开发针对1918 NS1的潜在抑制剂。通过一个 结合小角度X射线散射，NMR光谱，分子动力学的协同方法 模拟和基于细胞的暗示，我们将追求以下特定目标。目标1。确定结构 1918 NS1：CRKII相互作用的机理使用一系列生物物理实验。劫持和搬迁 CRKII进入核是1918年大流行IAV NS1的独特特征。要了解这个过程，我们 将揭示1918 NS1的亲和力和寿命（1/koff）的结构和能量机制：CRKII 复合物是调制的。目标2。确定NS1诱导的PI3K激活的基础机制。 1918 NS1：CRKII相互作用显着增强了NS1诱导的PI3K激活。但是，它的分子 机制是未知的。我们将寻求全面确定分子机制 1918 NS1：CRKII复合物与PI3K的p85亚基相互作用，揭示了其在PI3K激活中的功能作用，并且 识别Hotspot NS1保留与CRKII和p85相互作用的。这项研究有望具有阳性 对靶向NS1宿主蛋白相互作用的抗病毒剂的发展的影响。