RESUMEN
Seasonal epidemics and periodic worldwide pandemics caused by influenza A viruses are of continuous concern. The viral nonstructural (NS1) protein is a multifunctional virulence factor that antagonizes several host innate immune defenses during infection. NS1 also directly stimulates class IA phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. Here, we present a 2.3-A resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85beta, a regulatory subunit of PI3K. Our data emphasize the remarkable isoform specificity of this interaction, and provide insights into the mechanism by which NS1 activates the PI3K (p85beta:p110) holoenzyme. A model of the NS1:PI3K heterotrimeric complex reveals that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts between the N-terminal SH2 domain of p85beta and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic alpha-helix of NS1 sits adjacent to the highly basic activation loop of the enzyme. During infection, a recombinant influenza A virus expressing NS1 with charge-disruption mutations in this acidic alpha-helix is unable to stimulate the production of phosphatidylinositol 3,4,5-trisphosphate or the phosphorylation of Akt. Despite this, the charge-disruption mutations in NS1 do not affect its ability to interact with the p85beta inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85beta (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/metabolismo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Animales , Secuencia de Bases , Dominio Catalítico , Bovinos , Línea Celular , Cristalografía por Rayos X , Cartilla de ADN/genética , Perros , Activación Enzimática , Humanos , Técnicas In Vitro , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Modelos Moleculares , Complejos Multiproteicos , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Proteínas no Estructurales Virales/genética , Dominios Homologos srcRESUMEN
Posttranslational modification of viral proteins by cellular enzymes is a feature of many virus replication strategies. Here, we report that during infection the multifunctional human influenza A virus NS1 protein is phosphorylated at threonine-215. Substitution of alanine for threonine at this position reduced early viral propagation, an effect apparently unrelated to NS1 antagonizing host interferon responses or activating phosphoinositide 3-kinase signaling. In vitro, a subset of cellular proline-directed kinases, including cyclin dependent kinases (CDKs) and extracellular signal-regulated kinases (ERKs), potently phosphorylated NS1 protein at threonine-215. Our data suggest that CDK/ERK-mediated phosphorylation of NS1 at threonine-215 is important for efficient virus replication.
