Structure and functional dynamics of virus-host protein interactions

1918年甲型流感病毒(IAV)，也被称为西班牙流感，导致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，与抑制抗病毒相关。 病毒免疫反应，因此已被确定为高水平的分子决定因素之一 1918年IAV的致病性。1918年IAV的NS1(1918NS1)包含一个富含脯氨酸的基序(PRM)，它介导 与宿主CrkII结合具有高亲和力和选择性。1918年NS1：CrkII的相互作用在 抑制宿主抗病毒免疫反应，增强病毒复制。此外，NS1的 许多禽类/猪IAV都含有与CrkII结合的PRM。鉴于IAV的人畜共患病潜力，有一个关键的 需要确定1918NS1和细胞CrkII相互作用的分子机制。 我们研究计划的长期目标是阐明病毒宿主的分子机制。 蛋白质的相互作用。我们在这项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年NS1：CrkII的分子和细胞机制 复合体诱导PI3K激活，导致病毒复制增强。我们的中心假设是1918年 Ns1：CrkII复合体在结构上是动态的，这对于与P85的相互作用具有重要的功能 PI3K调节亚基。为了验证这一假设，我们将确定1918年NS1：CrkII复合体的结构 并阐明该复合体如何与P85相互作用激活PI3K信号通路。我们对此的理由是 研究表明，从机制上理解1918年ns1与CrkII和p85的相互作用将有所帮助。 确定以前未发现的靶点，以开发针对1918年NS1的潜在抑制剂。通过一个 结合小角X射线散射、核磁共振光谱、分子动力学的协同方法 模拟和基于细胞的分析，我们将追求以下具体目标。目标1.确定结构 用生物物理实验电池研究1918年ns1：crkII相互作用的机制。劫持和重新安置 CrkII进入细胞核是1918年IAV NS1大流行的一个显著特征。为了了解这一过程，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 复数是调制的。目的2.探讨NS1诱导PI3K活化的分子机制。 1918年NS1：CrkII相互作用显著增强NS1诱导的PI3K激活；但其分子 机制尚不清楚。我们将寻求全面确定分子机制，其中 1918NS1：CrkII复合体与PI3K的P85亚基相互作用，揭示了它在PI3K激活中的功能作用，以及 确定与crkII和p85相互作用的热点ns1残基。这项研究预计将有积极的 对针对NS1-宿主蛋白相互作用的抗病毒药物的开发的影响。

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.