NS1 (nonstructural protein 1) is an important virulence factorPandemic as well as seasonal outbreaks of influenza A virus represent major threats to global public health. In the last century three major pandemics have occurred, in 1918, 1957, and 1968, caused by H1N1 (Spanish flu), H2N2 (Asian flu), and H3N2 (Hong Kong flu) viruses, respectively. Of these, the Spanish flu was the most severe and is estimated to have caused over 40 million deaths worldwide (1). Recent human infections by highly pathogenic H5N1 avian influenza A viruses have increased the concern that another global pandemic may occur.Influenza A virus belongs to the Orthomyxoviridae family of enveloped viruses. Its genome is organized into eight singlestranded, negative-sense RNA segments that code for 11 identified viral proteins (2). NS1 (nonstructural protein 1) is encoded by the shortest RNA segment 8. It is expressed early in viral replication cycle, and it is not a component of the virus particle (2). Instead, NS1 is a multifunctional virulence factor that promotes virus replication in the host cell and helps to evade antiviral immunity (3-5). In particular, NS1 uses several mechanisms to prevent suppression of influenza A virus replication by the type I interferon system of the host.Recent studies have demonstrated that during influenza A virus infection NS1 protein activates the phosphatidylinositol 3-kinase (PI3K) 3 signaling pathway, apparently via its association with the p85 regulatory subunit of PI3K (6 -9). Activation of the PI3K pathway seems to be important for influenza A virus replication, because in cell culture studies recombinant viruses with mutations that prevented binding of NS1 to p85 formed much smaller plaques and grew to 10-fold lower titers than the wild-type virus (9). Moreover, compounds that inhibit PI3K can strongly suppress influenza A virus replication (7, 9, 10).Hale et al. (9) showed that the tyrosine residue 89 (Tyr-89) of NS1 protein serves a critical role in mediating binding to p85␤. This tyrosine lies in the context similar to a YXNM motif, which upon tyrosine phosphorylation can serve as a high affinity binding site for the SH2 domain of p85 (11), but apparently NS1 interacts with p85␤ in an SH2-independent manner (12). In addition, p85 contains an SH3 domain, and Zhou and co-workers (6, 13) have suggested that a PXXP sequence in NS1, which resembles the consensus of an SH3-binding motif (see below), may also contribute to the p85 interaction.SH3 domains are small protein modules that mediate interand intramolecular protein interactions and are often found in proteins regulating cellular signaling pathways, cytoskeletal organization, and membrane trafficking (14, 15). SH3 domains recognize short proline-rich sequences, which are typically characterized by (ϩ)-X⌽PXXP (class I) or PX⌽PX-(ϩ) (class II) consensus sequences (where X is any amino acid; (ϩ) indicates a positively charged residue; and ⌽ indicates a hydrophobic residue) (15)(16)(17). Since the discovery that the human immunodeficiency virus type...