Dr Rupert Russell, University of St Andrews
Seasonal epidemics and worldwide pandemics caused by influenza A viruses are of continuous public health concern. During infection, the viral nonstructural (NS1) protein stimulates phosphoinositide 3-kinase (PI3K) signaling, an essential cell survival pathway commonly mutated in human cancers. A structure of the NS1 effector domain in complex with the p85β inter-SH2 (coiled-coil) domain suggests that NS1 uses the coiled-coil as a structural tether to sterically prevent normal inhibitory contacts within the PI3K (p85β:p110) holoenzyme. Intriguingly, a small acidic α-helix of NS1 also sits adjacent to the highly basic activation loop of p110, plausibly hijacking p110 catalytic activity directly. Biochemically, we found that charge-disruption mutations in this α-helix completely abrogate NS1- mediated PI3K activation, yet do not affect NS1 binding the inter-SH2 domain. Thus, our data expand the structural and molecular understanding of aberrant p110 activation, and imply that similar novel regulatory mechanisms may occur during normal homeostatic control of PI3K.
Class IA phosphoinositide 3-kinases (PI3Ks) are obligate heterodimeric enzymes consisting of a 110 kDa catalytic subunit (p110α, p110β, or p110 γ) bound to a non-catalytic 85 kDa regulatory subunit (typically p85a or p85β) . Growth factor receptor-mediated activation of PI3K requires the relocalisation of p85:p110 heterodimers to the plasma membrane, where disinhibition of p110 by p85 leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 is an intracellular lipid second messenger that Diamond Light Source Annual Report 2010 recruits pleckstrin homology domain-containing protein kinases (such as Akt) to the membrane. Subsequent activation of these kinases stimulates a plethora of signalling cascades that regulate diverse biological processes, including cell survival, proliferation, and metabolism .
During infection, the PI3K signalling pathway is activated by influenza A viruses in order to promote efficient virus replication [3,4]. Specifically, the multifunctional viral NS1 protein binds directly to p85β, but not p85a, and stimulates the lipid kinase activity of p85β-associated p110 [4-7]. The p85β regulatory subunit is made up of five domains: an N-terminal SH3 domain, a GTP-ase activating protein (GAP) domain, and two SH2 domains (termed nSH2 and cSH2), which flank the inter-SH2 (β-iSH2) domain. NS1 consists of an N-terminal RNA-binding domain (RBD) flexibly linked to a C-terminal ‘effector’ domain (ED) [6, 8]. We have previously shown that β-iSH2 (a rigid coiled-coil scaffold for the p110 subunit) is the primary direct binding site for the NS1 ED . In order to gain further insights into how NS1 might modify normal inter- and intra- molecular regulatory contacts within the p85β:p110 holoenzyme, we purified and crystallised the ED:β-iSH2 complex. The structure was solved to 2.3 Å using data collected at Diamond on beamline I03.
Fig.1 Surface representation of the heterodimeric
The heterodimeric ED:β-iSH2 structure thus resembles a ‘golf-club’ shaped complex (Fig. 1). Central to the complex is the formation of a 4-helix bundle composed of a short conserved acidic α-helix of NS1 ED (residues 95-100) and all three a-helices of β-iSH2. NS1 ED binds to the end of β-iSH2 opposite the major p110 interaction site, and encompasses residues 562-589 of p85β. Such positioning of NS1 ED is in full agreement with previous β-iSH2 deletion mapping studies.
Despite high sequence identity between the two p85 iSH2 domains, it is remarkable that NS1 binds only to the p85β, but not p85a, isoform of PI3K. Previous mutational studies on β-iSH2 clearly defined Val573 as critically mediating the NS1:p85β interaction. Reciprocal gain of function experiments with p85a further demonstrated that mutation of Met582 (the corresponding residue of p85a) to valine allows NS1 to bind this mutant p85a and subsequently stimulate the lipid kinase activity of p85a-associated p110. In the ED:β-iSH2 complex presented here, Val573 lies directly at the interface and buries over 70Å2 upon complex formation. It is clear that a methionine at this position would be sterically precluded, and serves to explain structurally the p85 isoform specificity exhibited by NS1.
To investigate possible mechanisms for NS1-mediated activation of PI3K, we superimposed our ED:-iSH2 structure onto the reported crystal structure of full-length p110α in complex with p85a iSH2 by aligning the two homologous iSH2 domains (Fig. 2). Remarkably, there is almost no overlap between the Ca chains of p110α and NS1 ED in this model, suggesting that both complex structures are compatible with one another, and that a heterotrimeric NS1:p85β:p110 complex can occur physiologically. Surprisingly, in addition to physically preventing the inhibitory action of p85β nSH2, our model suggests that the NS1 ED may also directly influence the kinase activity of p110. The activation loop of p110 (residues 933-957) lies close to the proposed interface of the p110 kinase domain with NS1 ED. In the published crystal structure of the iSH2:p110α complex, residues 941-950 (KKKKFGYKRE) are disordered possibly reflecting an inactive state of the activation loop. In our model, the small acidic α-helix of NS1 ED that participates in the 4-helix bundle of the ED:β-iSH2 complex sits adjacent to this section of the highly basic activation loop, and therefore has the potential to interact with it
|Fig. 2. Structural model of the heterotrimeric NS1:p85β:p110 complex, highlighting the potential interaction between NS1 ED and the kinase activation loop.|
The structural basis for influenza A virus NS1 protein binding p85β and activating PI3K expands our current knowledge on this important multifunctional (and ‘multistructural’) virulence factor. Although NS1 binding to the β-iSH2 domain will disrupt the proposed inhibitory contacts between p85β nSH2 and p11010, this does not appear sufficient to activate PI3K. We therefore propose that to stimulate kinase activity the acidic α-helix of NS1 must also directly interact with the p110 activation loop. These insights into the structural and functional interactions of NS1 with PI3K could lead to the rational design of novel therapeutics, and underscore the multidisciplinary importance of studying virus-host interactions.
 L. C. Cantley, Science, 296, 1655 (2002).
 B. D. Manning, L. C. Cantley, Cell, 129, 1261 (2007).
 C. Ehrhardt et al., J Virol 81, 3058 (2007).
 B. G. Hale, D. Jackson, Y. H. Chen, R. A. Lamb, R. E.Randall, Proc Natl Acad Sci USA, 103, 14194 (2006).
 Y. Li, D. H. Anderson, Q. Liu, Y. Zhou, J Biol Chem, 283, 23397 (2008).
 B. G. Hale, R. E. Randall, J. Ortin, D. Jackson, J Gen Virol, 89, 2359 (2008).
 B. G. Hale, I. H. Batty, C. P. Downes, R. E. Randall, J Biol Chem, 283, 1372 (2008).
 Z. A. Bornholdt, B. V. Prasad, Nature, 456, 985 (2008).
Principal Publications and Authors
BG Hale, PS Kerry, D Jackson, BL Precious, A Gray, MJ Killip, RE Randall, RJ Russell; Structural insights into phosphoinositide 3-kinase activation by the influenza A virus NS1 protein; Proceedings of the National Academy of Sciences USA; 107:1954-1959 (2010).
Medical Research Council, U.K. and Scottish Funding Council.
Diamond Light Source is the UK's national synchrotron science facility, located at the Harwell Science and Innovation Campus in Oxfordshire.
Copyright © 2017 Diamond Light Source
Diamond Light Source Ltd
Harwell Science & Innovation Campus