Structure and functional dynamics of virus-host protein interactions

1918年的甲型流感病毒（IAV），也被称为西班牙流感，引起了人类最严重的流感大流行 历史非结构蛋白1（NS1）是一种多功能的毒力因子，与抗- 病毒免疫应答，因此已被鉴定为高免疫应答的分子决定因素之一。 1918年IAV的致病性1918年IAV的NS1（1918 NS1）含有一个富含脯氨酸的基序（PRM），其介导 以高亲和力和选择性与宿主CrkII结合。1918年NS1：CrkII相互作用在 抑制宿主抗病毒免疫应答和增强病毒复制。此外， 许多禽类/猪IAV含有CrkII结合PRM。考虑到IAV的人畜共患病潜力， 需要确定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相互作用的分子和细胞机制。 复合物诱导PI3K活化，导致增强的病毒复制。我们的核心假设是，1918年 NS1：CrkII复合物是结构动态的，其在与p85蛋白相互作用中具有重要的功能。 PI3K调节亚基。为了验证这一假设，我们将确定1918年NS1：CrkII复合物的结构。 并阐明该复合物如何与p85 β相互作用以激活PI3K信号通路。我们的理由 研究表明，1918 NS1与CrkII和p85 β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 结合小角X射线散射、NMR光谱、分子动力学的协同方法 模拟和基于细胞的测定，我们将追求以下具体目标。目标1。为了确定结构 1918年NS1：CrkII相互作用的机制，使用生物物理实验电池。劫持和迁移 CrkII进入细胞核是1918年大流行IAV NS 1的显著特征。为了理解这个过程，我们 将揭示1918年NS1：CrkII的亲和力和寿命（1/koff）的结构和能量机制 复合物被调制。目标二。确定NS1诱导PI3K活化的分子机制。 1918 NS1：CrkII相互作用显著增强NS1诱导的PI3K活化;然而，其分子生物学效应不显著。 机制不明。我们将寻求全面确定的分子机制， 1918 NS1：CrkII复合物与PI3K的p85 β亚基相互作用，揭示了其在PI3K活化中的功能作用， 鉴定与CrkII和p85 β相互作用热点NS1残基。这项研究预计将有一个积极的 对靶向NS1-宿主蛋白相互作用的抗病毒药物的发展的影响。

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.