Structure and functional dynamics of virus-host protein interactions

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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.