Mechanism of influenza A virus NS1 protein interaction with the p85beta, but not the p85alpha, subunit of phosphatidylinositol 3-kinase (PI3K) and up-regulation of PI3K activity.
Influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt pathway by binding influenza A virus NS1 protein to the p85beta regulatory subunit of PI3K. In this study, we report that NS1 binds to the inter-SH2 (iSH2) domain of p85beta. Mutational analyses on p85beta iSH2 domain defined that Val-573 is the critical amino acid (AA) that mediates NS1 and p85beta interaction. In reciprocal gain of function experiments with p85alpha, we demonstrated that mutation to Val at Met-582 leads to NS1 binding and increased PI3K activity. Molecular modeling based on our experimental results suggested that, in addition to the interaction interface between the NS1 SH3 binding motif 1 (AA 164-167) and p85beta Val-573, AA 137-142 in NS1 might interact with p85beta. Indeed, mutations of AA 141 and 142 in NS1 disrupted the interaction between NS1 and p85beta. Mutant virus PR8-NS1-141/142 was not able to activate Akt phosphorylation. Furthermore, PI3K assays demonstrated that, in wild-type virus-infected cells, p85beta-associated PI3K activity was increased significantly. In contrast, in the mutant virus-infected cells containing mutant NS1 unable to interact with p85beta, the p85beta-associated PI3K activity up-regulation was not seen, suggesting that PI3K up-regulation is dependent upon the interaction between NS1 and p85beta. Competition experiments and the immunoprecipitation studies demonstrated that NS1, p85beta, and p110 form a complex in cells. Finally, the mechanism by which binding of NS1 to p85beta regulates PI3K activity was discussed based on a predicted structural model of NS1-p85-p110 complex.
Li
Yang
Y
Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E3, Canada.
Anderson
Deborah H
DH
Liu
Qiang
Q
Zhou
Yan
Y
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D009980:Influenza A virus / Q000378:metabolism; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015854:Up-Regulation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000502:physiology*; D018909:src Homology Domains
C085820:INS1 protein, influenza virus; D017361:Viral Nonstructural Proteins; D019869:Phosphatidylinositol 3-Kinases