Influenza A virus NS1 protein binds p85␤andactivates phosphatidylinositol-3-kinase signalingBenjamin G. Hale*, David Jackson†, Yun-Hsiang Chen*, Robert A. Lamb†‡, and Richard E. Randall*‡*Centre for Biomolecular Sciences, University of St. Andrews, St. Andrews, Fife KY16 9ST, United Kingdom; and†Howard Hughes Medical Instituteand Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208-3500Contributed by Robert A. Lamb, July 25, 2006Influenza A virus NS1 is a multifunctional protein, and in virus-infected cells NS1 modulates a number of host-cell processes byinteracting with cellular factors. Here, we report that NS1 bindsdirectly to p85␤, a regulatory subunit of phosphatidylinositol-3-kinase (PI3K), but not to the related p85␣subunit. Activation ofPI3K in influenza virus-infected cells depended on genome repli-cation, and showed kinetics that correlated with NS1 expression.Additionally, it was found that expression of NS1 alone wassufficient to constitutively activate PI3K, causing the phosphory-lation of a downstream mediator of PI3K signal transduction, Akt.Mutational analysis of a potential SH2-binding motif within NS1indicated that the highly conserved tyrosine at residue 89 isimportant for both the interaction with p85␤, and the activation ofPI3K. A mutant influenza virus (A兾Udorn兾72) expressing NS1 withthe Y89F amino acid substitution exhibited a small-plaque pheno-type, and grew more slowly in tissue culture than WT virus. Thesedata suggest that activation of PI3K signaling in influenza Avirus-infected cells is important for efficient virus replication.Akt phosphorylation 兩 multifunctional NS1 protein 兩 reverse geneticsInfluenza A viruses are globally important human and animalrespiratory pathogens that are responsible for both seasonal‘‘flu’’ outbreaks, and periodic world-wide pandemics (1). Themultifunctional NS1 protein of influenza A is widely regarded asa virulence factor (2), and contributes significantly to diseasepathogenesis by modulating many virus and host-cell processes(3–5). A major role of NS1 is as a type I IFN antagonist: NS1down-regulates host innate IFN-mediated antiviral responsesduring infection (3–5). To perform such activities, NS1 func-tionally interacts with virus- and兾or cell-derived factors: e.g.,NS1 blocks the activation of two IFN-inducible antiviral pro-teins: 2⬘-5⬘-oligoadenylate synthetase (2⬘-5⬘ -OAS), and thedsRNA-dependent protein kinase R (PKR). In infected cells, thebinding of NS1 to dsRNA (a putative by-product of viral RNAgenome replication) has been implicated in the inhibition ofcellular 2⬘ -5⬘-OAS (6). dsRNA-binding by NS1 may also be arequirement for blocking the activation of PKR in vitro (7), butstudies now suggest that this inhibition, both in vitro and in vivo,may occur via direct interaction of NS1 with PKR (8, 9).The biological activities of NS1 are likely to be strain- and兾orcell-type specific (10). For influenza virus A兾Udorn兾72 (Ud), ithas been shown that NS1 interacts with two cellular proteinsinvolved in mRNA processing and transport: the 30-kDa subunitof the cleavage and polyadenylation specificity factor (CPSF30),and poly(A)-binding protein II (PABII) (11, 12). The directbinding of Ud兾NS1 to these two proteins blocks the posttran-scriptional processing of the 3⬘ ends of cellular pre-mRNAs,inhibiting nucleo-cytoplasmic mRNA export, and limiting bothIFN-␤production and the host response to IFN (11–14). How-ever, studies with influenza virus A兾Puerto Rico兾8兾34 (PR8)suggest that during infection NS1 blocks the pretranscriptionalnuclear translocation of both IRF-3 and NF-␬B (thus limitingIFN-␤promoter activation), possibly by binding and sequester-ing dsRNA away from intracellular sensors (15, 16). Interest-ingly, PR8兾NS1 was unable to block the activation of an IFN-stimulated response element (ISRE) reporter construct inresponse to exogenous IFN-␣(10). In contrast, the NS1 proteinsof other influenza virus strains, including A兾Victoria兾3兾75(Vic), efficiently limit ISRE activation probably by blocking aposttranscriptional process (10). These data suggest that, unlikeother NS1 proteins, PR8兾NS1 is incapable of inhibiting theposttranscriptional processing of cellular pre-mRNAs, and thusmay limit the synthesis of IFN-␤by a pretranscriptional mech-anism that is distinct from the posttranscriptional processadopted by many other strains.We generated a HEp2 cell line that constitutively expressesPR8兾NS1 as part of our studies on viral evasion of host innateimmunity. It was found that PR8兾NS1 binds specifically to thep85␤regulatory subunit of phosphatidylinositol-3-kinase(PI3K), and subsequently activates PI3K signaling. These obser-vations were extended by finding that Ud兾NS1 and Vic兾NS1 alsospecifically bind p85␤. PI3K is a heterodimeric protein兾lipidkinase that consists of a regulatory subunit (usually p85␣, p85␤,or p55␥), and a p110 catalytic subunit (␣,␤,␥,or␦) (17).Activation of PI3K causes the generation of the second messen-ger phosphatidylinositol (3,4,5)-triphosphate, which acts to re-cruit pleckstrin homology domain-containing proteins (such asthe key downstream mediator of PI3K, Akt), to membranes,where they are further activated by phosphorylation (18–20).The modulation of host-cell PI3K signaling is a target for manyacute- and chronic- disease-causing viruses, and induces a num-ber of physiological changes within cells that aid virus replication(i.e., regulation of virus兾host gene transcription, protein synthe-sis, or cell survival) (21). We observed that activation of PI3Kalso aids the replication of influenza A virus. By using reversegenetics, a mutant influenza virus (Ud strain) was generated thatcontains an amino acid substitution in NS1 at the highly con-served tyrosine residue 89. This mutant is unable to bind p85␤or activate PI3K signaling, and forms smaller plaques and growsless efficiently than WT Ud influenza virus.ResultsThe NS1 Protein of Influenza A Virus Interacts with p85␤. A HEp2 cellline was isolated that constitutively expresses a V5 epitope-tagged form of PR8兾NS1, an influenza virus protein that hasbeen reported to limit both the production and downstreameffects of IFN (3, 4). Characterization of the PR8兾NS1-expressing cell line confirmed previously observed NS1 func-tions: NS1 limits PKR activation in response to virus