Abstract
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.
Highlights
Mutant viruses containing Src homology 3 (SH3) motif 1 mutation in NS1 cannot bind to p85, and are unable to activate the phosphatidylinositol 3-kinase (PI3K)/ Akt pathway
NS1 Binds to the iSH2 Domain of p85—We and others have To identify the region of p85 that interacts with NS1, different previously shown that the influenza A virus NS1 protein inter- domains of p85 were fused to glutathione S-transferase (GST) (Fig. 1A)
In virus-infected cells the ratio of p85␣/p85pan and ulation of PI3K Activity—Our previous studies showed that p85/p85pan did not change dramatically compared with that influenza A virus infection led to Akt phosphorylation, which was PI3K-dependent [24], and mutant viruses PR8-SH3-mf-1 and PR8-NS1–141/142, whose NS1 proteins did not interact with p85, could not activate the PI3K/Akt pathway (Fig. 6C) [22]
Summary
Cells and Virus—A549 cells (human lung carcinoma cells) and 293T (human embryonic kidney) cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen) containing 10% fetal bovine serum. For glutathione S-transferase (GST) fusion protein constructs, cDNA fragments corresponding to each domains of mouse p85 SH3 (AA 1–106), BCR (AA 82–322), nSH2 (AA 297– 432), iSH2 (AA 420 – 615), and cSH2 (AA 598 –722) were PCR-amplified from pGEX-4T3-p85 and cloned into pGEX5X-1 (GE Healthcare) at EcoRI/XhoI sites. The precipitated proteins were subjected to either SDS-PAGE followed by Western blotting with specific antibodies or PI3K assay. To calculate the potential interaction domains between p85 iSH2 and NS1, crystal coordinates of p85 iSH2 (Protein Data Bank code 2V1Y) was used as the templates, after manual replacement of Met582 in p85␣ by Val as suggested by our experiments described in the text, the local structure of the mutated residue and its neighbors was optimized using the MODELER program. Marker mock 10% input GST SH3 BCR nSH2 iSH2 cSH2 p85β marker mock 10% input GST SH3 BCR nSH2 iSH2 cSH2 p85β
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