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-Å resolution crystal structure of the NS1 effector domain in complex with the inter-SH2 (coiled-coil) domain of p85β, 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 (p85β: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 p85β and the p110 catalytic subunit. Furthermore, in this model, NS1 makes extensive contacts with the C2/kinase domains of p110, and a small acidic α-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 α-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 p85β inter-SH2 domain in vitro. Overall, these data suggest that both direct binding of NS1 to p85β (resulting in repositioning of the N-terminal SH2 domain) and possible NS1:p110 contacts contribute to PI3K activation.C lass IA phosphoinositide 3-kinases (PI3Ks) are obligate heterodimeric enzymes consisting of a 110-kDa catalytic subunit (p110α, p110β, or p110δ) bound to a noncatalytic 85-kDa regulatory subunit (typically p85α or p85β) (1). Growth factor receptor-mediated activation of PI3K requires the relocalization of p85:p110 heterodimers to the plasma membrane, where disinhibition of p110 by p85 leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). PIP 3 is an intracellular lipid second messenger that recruits pleckstrin homology domain-containing effectors (including protein kinases such as Akt) to the membrane. Subsequent activation of these effectors stimulates a plethora of signaling cascades that regulate diverse biological processes, including cell survival, proliferation, and metabolism (2). Given that PI3K is among the most frequently mutated enzymes associated with human cancers (3, 4), there is considerable interest in trying to understand the structural basis for both normal and pathophysiological regulation of p110 by p85 (5-7). Such studies are likely to yield insights into the novel mechanisms by which PI3K can be aberrantly activated, and may provide the focus for designing selective inhibitors targeting specific diseases.During infection, the PI3K signaling pathway is activated by influenz...

The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.Simian virus type 5 (SV5) is classified within the genus Rubulavirus of the subfamily Paramyxovirinae of the family Paramyxoviridae (18). It is now well established that most members of the Paramyxovirinae subfamily at least partially circumvent the interferon (IFN) response by blocking IFN signaling and reducing the production of IFN by infected cells (for reviews see (1,10,13,22,32). In human cells, SV5 blocks both IFN-␣/␤ and IFN-␥ signaling by targeting STAT1 (a transcription factor which is essential for IFN signaling) for proteasome-mediated degradation (3,7,23,25,36,37). The molecular mechanisms by which SV5 targets STAT1 for degradation have been the subject of several recent investigations, and of the virus proteins, only the V protein is required to mediate this process (3,7,26).The SV5 V protein is the 222-amino-acid product of a faithful mRNA copy of the second open reading frame (the V/P gene) of the virus genome. V has been shown to be a multifunctional protein that, apart from its involvement in STAT1 degradation, also interacts with an IFN-inducible DExD/H box helicase mda-5 to limit the production of IFN (1), binds singlestranded RNA (20) and may act as a chaperone keeping the nucleoprotein of the virus soluble (28). STAT1 degradation, mediated by SV5 V protein, is independent of IFN signaling or phosphorylation of STAT proteins (3, 24). However, there is an absolute requirement for STAT2 in STAT1 degradation and consequently, SV5 infection fails to induce the degradation of STAT1 in STAT2-deficient (U6A) cells (26) or in 2fTGH cells that ex...

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.

In vitro and in vivo specificity of ubiquitination and degradation of STAT1 and STAT2 by the V proteins of the paramyxoviruses simian virus 5 and human parainfluenza virus type 2 Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous. well-established that SV5 and hPIV2, and many other paramyxoviruses, at least partially circumvent the IFN response by blocking IFN signalling and IFN production (reviewed by Garcia-Sastre, 2004;Horvath, 2004;Nagai & Kato, 2004). In human cells, SV5 and mumps virus block IFN signalling by targeting STAT1 for proteasomemediated degradation, whilst human hPIV2 targets STAT2 for degradation (Andrejeva et al., 2002b;Didcock et al., 1999a;Nishio et al., 2001;Parisien et al., 2001;Yokosawa et al., 2002;Young et al., 2000). As a consequence, SV5 inhibits both IFN-a/b and IFN-c signalling, whilst, in human cells, hPIV2 only blocks IFN-a/b signalling. Intriguingly, it has recently been reported that mumps virus, but not SV5, can also target STAT3 for degradation, although the biological significance of this has yet to be established (Ulane et al., 2003). The importance of IFN in controlling SV5 infections can be judged from studies in mice: SV5 fails to degrade STAT1 in murine cells and is non-pathogenic in normal and severe combined immunodeficient mice (which fail to make an adaptive immune response; Didcock et al., 1999b;Randall & Young, 1991) INTRODUCTION

SUMMARYThe ability of adenovirus structural polypeptides to bind nucleic acids was assessed by separating the polypeptides on SDS-polyacrylamide gels, transferring them electrophoretically to nitrocellulose and probing with 32p-labelled nucleic acids. Polypeptides IVa2, V, VI and VII, as well as trace amounts of pVII and a polypeptide of apparent mol. wt. 40 x 103 were able to bind label under these conditions. Labelling was also detected with a smaller polypeptide, possibly related to the cleavage products of pVII and/or pVI. The binding of DNA to polypeptide VI appeared to be more sensitive to detergents than the others. No sequence specificity could be detected in the DNA binding.