The Structure Basis Of Interaction Between Influenza A Virus Non-Structure Protein NS1 And PI3K Regulatory Subunit P85β Protein

Phosphatidylinositol-3-kinase(PI3K) is a key signaling mediator that is activated by Influenza A virus.In fact,PI3K not only regulates a very early step during viral entry but also results in suppression of premature apoptosis at later stages of infection. The latter function is dependent on the expression of the viral non-structural protein-1 (A/NS1).It has been shown that PI3K activation occurs by direct interaction of A/NS1 with the P85βregulatory subunit and interaction sites of A/NS1 and P85βhave now been mapped in detail,that are the effector domain of NS1 protein and the iSH2 and SH3 domains of the P85βprotein.However,the way of how the PI3K is activated by the direct binding of NS1 with the P85βremains unclear.Thus,obtaining the NS1-P85βprotein complex crytals so as to locate the accurate binding sites of the two proteins in 3D space would be an effective potential way to unravel the PI3K activation mechanism upon NS1 and P85βbinding.In our experiment,we construct and express the H5N1 NS1 effector domain as well as the P85βiSH2 and SH3 domains clones,then repeat the interaction related domain mapping experiment.Our result shows that only P85βiSH2 directly interacts with NS1 protein,while SH3 domain of P85βseems to fail to bind to NS1 protein. After verifying the interaction does not occur upon strain specific,we then tried a series of biochemical methods to purify the NS1 and P85βprotein as well as their hetero-dimer Complex.We then use the vapor diffusion method to screen the protein crystal growth conditions,and now the work is still in progress.