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. 2007 Dec;81(23):12730-9.
doi: 10.1128/JVI.01427-07. Epub 2007 Sep 19.

SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signaling pathway activation

Yeun-Kyung Shin  1 , Yang Li, Qiang Liu, Deborah H Anderson, Lorne A Babiuk, Yan Zhou
Affiliations

SH3 binding motif 1 in influenza A virus NS1 protein is essential for PI3K/Akt signaling pathway activation

Yeun-Kyung Shin et al. J Virol. 2007 Dec.

Abstract

Recent studies have demonstrated that influenza A virus infection activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by binding of influenza NS1 protein to the p85 regulatory subunit of PI3K. Our previous study proposed that two polyproline motifs in NS1 (amino acids 164 to 167 [PXXP], SH3 binding motif 1, and amino acids 213 to 216 [PPXXP], SH3 binding motif 2) may mediate binding to the p85 subunit of PI3K. Here we performed individual mutational analyses on these two motifs and demonstrated that SH3 binding motif 1 contributes to the interactions of NS1 with p85beta, whereas SH3 binding motif 2 is not required for this process. Mutant viruses carrying NS1 with mutations in SH3 binding motif 1 failed to interact with p85beta and induce the subsequent activation of PI3K/Akt pathway. Mutant virus bearing mutations in SH3 binding motif 2 exhibited similar phenotype as the wild-type (WT) virus. Furthermore, viruses with mutations in SH3 binding motif 1 induced more severe apoptosis than did the WT virus. Our data suggest that SH3 binding motif 1 in NS1 protein is required for NS1-p85beta interaction and PI3K/Akt activation. Activation of PI3K/Akt pathway is beneficial for virus replication by inhibiting virus induced apoptosis through phosphorylation of caspase-9.

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Figures

FIG. 1.
FIG. 1.
Characterization of mutant viruses. (A) Schematic diagram showing the locations of two SH3 binding motifs on WT NS1 and the changes in amino acid sequence in NS1 encoded by mutant viruses. (B) Plaques formed by WT, PR8-SH3-mf-1, PR8-SH3-mf-2, and PR8-SH3-mf-1/2 viruses on MDCK cells. (C) Multiple cycle growth curves of WT, PR8-SH3-mf-1, PR8-SH3-mf-2, and PR8-SH3-mf-1/2 on MDCK cells. Cells were infected in triplicate with each virus at an MOI of 0.001. Media were collected at 12-h intervals until 72 h p.i., and titers were determined by plaque assay on MDCK cells. The mean titer values at each time point were plotted with the associated standard deviation displayed as error bars. (D) Intracellular localization of NS1 protein encoded by WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 mutant viruses. Cells were infected by the viruses at an MOI of 1; at 7 h p.i. the cells were fixed, permeabilized, and stained with NS1 antibody, followed by Cy2-conjugated anti-rabbit IgG.
FIG. 2.
FIG. 2.
SH3 binding motif 1 in NS1 is required for PI3K/Akt activation. A549 cells (A, B, and E) or MDCK cells (C and D) were mock, WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 infected at an MOI of 1 (A, B, C, and D) or at an MOI as indicated (E). Cell lysates prepared at 8 (A, C, and E) or 16 h (B and D) p.i. were subjected to Western blotting with phospho-Akt (Ser-473), phopho-Akt (Thr-308), Akt, or NS1 antibody.
FIG. 3.
FIG. 3.
SH3 binding motif 1 in NS1 is essential for NS1-p85β interaction in vitro. A549 cells were mock, WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 infected at an MOI of 1. (A) Cell lysates were prepared at 6 h p.i. 293T cells were transfected with pcDNA-3.1(−), pcDNA-NS1, pcDNA-NS1-SH3-mf-1, pcDNA-NS1-SH3-mf-2, or pcDNA-NS1-SH3-mf-1/2. (B) Cell lysates were prepared at 24 h posttransfection. GST-p85β or GST was immobilized to beads and incubated with the infected or transfected cell lysates. Precipitated proteins were subjected to either Western blotting with NS1 antibody or SDS-PAGE, followed by Coomassie blue staining.
FIG. 4.
FIG. 4.
SH3 binding motif 1 in NS1 is essential for NS1-p85β interaction in tissue culture. (A) 293T cells were cotransfected with either pcDNA3.1(−), pcDNA-NS1, pcDNA-NS1-SH3-mf-1, pcDNA-NS1-SH3-mf-2, or pcDNA-NS1-SH3-mf-1/2 each, together with pcDNA4HisMax-mp85β. As a negative control, pcDNA3.1(−), pcDNA-NS1, or its respective mutant plasmids were cotransfected with empty vector pcDNA4HisMaxB. At 24 h posttransfection, cell lysates were prepared and incubated with Ni-Sepharose beads. Precipitated proteins were subjected to Western blotting with NS1 or His6 antibody. Ten percent input was loaded as a control. (B) A549 cells were mock, WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 infected at an MOI of 1. Cell lysates were prepared at 6 h p.i., precleared by rabbit IgG-protein A, and incubated with either NS1 antibody-protein A or rabbit IgG-protein A. Precipitated proteins were subjected to Western blotting with NS1 or p85β antibody.
FIG. 5.
FIG. 5.
Cells infected by mutant viruses containing SH3 motif 1 mutation exhibit more severe cytopathic effect and morphological changes characteristic of apoptosis. MDCK cells were mock, WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 infected at an MOI of 1. At 16 h p.i., the morphology of the infected cells was either documented by light microscopy (A) or by immunofluorescent staining with M1 antibody and DAPI, followed by fluorescence microscopy (B). Arrows indicate the fragmented chromatin.
FIG. 6.
FIG. 6.
Influenza A virus induced pAkt phosphorylates caspase-9, leading to inhibition of apoptosis. MDCK cells were mock, WT, PR8-SH3-mf-1, PR8-SH3-mf-2, or PR8-SH3-mf-1/2 infected at an MOI of 1. At 16 (A) and 24 h (B) p.i., cell lysates were prepared and subjected to Western blotting with phospho-caspase-9 (Ser196) (A) or PARP (B) antibody, respectively. The total Akt level was monitored to verify equal loading of the samples.
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References

    1. Alessi, D. R., M. Andjelkovic, B. Caudwell, P. Cron, N. Morrice, P. Cohen, and B. A. Hemmings. 1996. Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15:6541-6551. - PMC - PubMed
    1. Bornholdt, Z. A., and B. V. Prasad. 2006. X-ray structure of influenza virus NS1 effector domain. Nat. Struct. Mol. Biol. 13:559-560. - PubMed
    1. Cardone, M. H., N. Roy, H. R. Stennicke, G. S. Salvesen, T. F. Franke, E. Stanbridge, S. Frisch, and J. C. Reed. 1998. Regulation of cell death protease caspase-9 by phosphorylation. Science 282:1318-1321. - PubMed
    1. Carpenter, C. L., K. R. Auger, M. Chanudhuri, M. Yoakim, B. Schaffhausen, S. Shoelson, and L. C. Cantley. 1993. Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit. J. Biol. Chem. 268:9478-9483. - PubMed
    1. Datta, S. R., A. Brunet, and M. E. Greenberg. 1999. Cellular survival: a play in three Akts. Genes Dev. 13:2905-2927. - PubMed

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