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Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein.

ABSTRACT: 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.

SUBMITTER: Hale BG

PROVIDER: S-EPMC2808220 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:NS1 of influenza A virus is a potent antagonist of host antiviral interferon responses. This multifunctional protein with two distinctive domains, an RNA-binding domain (RBD) and an effector domain (ED) separated by a linker region (LR), is implicated in replication, pathogenesis, and host range. Although the structures of individual domains of NS1 from different strains of influenza viruses have been reported, the only structure of full-length NS1 available to date is from an H5N1 strain (A/Vietnam/1203/2004). By carrying out crystallographic analyses of full-length H6N6-NS1 (A/blue-winged teal/MN/993/1980) and an LR deletion mutant, combined with mutational analysis, we show here that these full-length NS1 structures provide an exquisite structural sampling of various conformational states of NS1 that based on the orientation of the ED with respect to RBD can be summarized as "open," "semi-open," and "closed" conformations. Our studies show that preference for these states is clearly dictated by determinants such as linker length, residue composition at position 71, and a mechanical hinge, providing a structural basis for strain-dependent functional variations in NS1. Because of the flexibility inherent in the LR, any particular NS1 could sample the conformational space around these states to engage ED in different quaternary interactions so that it may participate in specific protein-protein or protein-RNA interactions to allow for the known multifunctionality of NS1. We propose that such conformational plasticity provides a mechanism for autoregulating NS1 functions, depending on its temporal distribution, posttranslational modifications, and nuclear or cellular localization, during the course of virus infection.NS1 of influenza A virus is a multifunctional protein associated with numerous strain-specific regulatory functions during viral infection, including conferring resistance to antiviral interferon induction, replication, pathogenesis, virulence, and host range. NS1 has two domains, an RNA-binding domain and an effector domain separated by a linker. To date, the only full-length NS1 structure available is that from an H5N1 strain (A/Vietnam/1203/2004). Here, we determined crystal structures of the wild type and a linker region mutant of the H6N6 NS1 (A/blue-winged teal/MN/993/1980), which together with the previously determined H5N1 NS1 structure show that NS1 exhibits significant strain-dependent structural polymorphism due to variations in linker length, residue composition at position 71, and a mechanical hinge. Such a structural polymorphism may be the basis for strain-specific functions associated with NS1.

Project description:The NS1 protein of influenza A virus is a multifunctional virulence factor that inhibits cellular processes to facilitate viral gene expression. While NS1 is known to interact with RNA and proteins to execute these functions, the cellular RNAs that physically interact with NS1 have not been systematically identified. Here we reveal a NS1 protein-RNA interactome and show that NS1 primarily binds intronic sequences. Among this subset of pre-mRNAs is the RIG-I pre-mRNA, which encodes the main cytoplasmic antiviral sensor of influenza virus infection. This suggested that NS1 interferes with the antiviral response at a posttranscriptional level by virtue of its RNA binding properties. Indeed, we show that NS1 is necessary in the context of viral infection and sufficient upon transfection to decrease the rate of RIG-I intron removal. This NS1 function requires a functional RNA binding domain and is independent of the NS1 interaction with the cleavage and polyadenylation specificity factor CPSF30. NS1 has been previously shown to abrogate RIG-I-mediated antiviral immunity by inhibiting its protein function. Our data further suggest that NS1 also posttranscriptionally alters RIG-I pre-mRNA processing by binding to the RIG-I pre-mRNA.IMPORTANCE A key virulence factor of influenza A virus is the NS1 protein, which inhibits various cellular processes to facilitate viral gene expression. The NS1 protein is localized in the nucleus and in the cytoplasm during infection. In the nucleus, NS1 has functions related to inhibition of gene expression that involve protein-protein and protein-RNA interactions. While several studies have elucidated the protein interactome of NS1, we still lack a clear and systematic understanding of the NS1-RNA interactome. Here we reveal a nuclear NS1-RNA interactome and show that NS1 primarily binds intronic sequences within a subset of pre-mRNAs, including the RIG-I pre-mRNA that encodes the main cytoplasmic antiviral sensor of influenza virus infection. Our data here further suggest that NS1 is necessary and sufficient to impair intron processing of the RIG-I pre-mRNA. These findings support a posttranscriptional role for NS1 in the inhibition of RIG-I expression.

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Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein.

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