Dimer Interface of the Effector Domain of Non-structural Protein 1 from Influenza A Virus

Abstract

Influenza is a contagious respiratory illness in humans caused by influenza A and B viruses that results in an annual average of 36,000 deaths in the United States alone (www.cdc. gov/Flu). Influenza A viruses, infecting birds and mammals, periodically cause widespread pandemics, the most deadly of which was the 1918 "Spanish Flu" pandemic that claimed an estimated 50 million lives (1). In recent years, the appearance of new H5N1 and H1N1 influenza A viruses, known as "avian flu" and "swine flu," respectively, has fueled fears of an impending deadly influenza pandemic in the 21st century. Moreover, the rapid emergence of influenza virus strains resistant to current antiviral drugs directed against influenza A neuraminidase and M2 ion channel accentuate the need for the development of new classes of influenza antivirals. Toward this end, several recent structural and functional studies of influenza proteins have illuminated new potential drug targets in influenza (2, 3).One such target is the non-structural protein 1 of influenza A, NS1A, a key multifunctional virulence factor produced in the host infected cell that plays a critical role in evading the host antiviral response (4). This highly conserved protein is composed of two domains, the 73-residue N-terminal doublestranded RNA-binding domain (RBD) 4 and the C-terminal (residues 86-end) effector domain (ED), joined by a flexible linker (Fig. 1). By binding non-specifically to dsRNA, the N-terminal RBD functions primarily to inhibit the interferon-induced 2Ј-5Ј oligonucleotide A synthetase/RNase L pathway (5). The C-terminal ED binds a plethora of host cellular proteins, including the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) (6, 7), the p85␤ subunit of phosphatidylinositol 3-kinase (PI3K) (8, 9), protein kinase R (PKR) (10, 11), and human tripartite motif 25, TRIM25, the E3 ubiquitin ligase of the retinoic acid-inducible gene I (RIG-I) (12, 13). All of these activities of NS1A ultimately contribute to the ability of influenza virus to suppress host responses to infection, including interferon production and apoptosis, critical to the life cycle of the virus. The recent developments of novel NS1 antagonists (14) and attenuated influenza viruses containing * This work was supported, in whole or in part, by National Institutes of Health Grant U54-GM094597 through the NIGMS Protein Structure Initiative (to G. T. M.) and National Institutes of Health Grants U01 AI074497 (to G. T. M. and R. M. K.) and R01 AI11772 (to R. M. K. 3 To whom correspondence may be addressed. E-mail: guy@cabm.rutgers.edu. 4 The abbreviations used are: RBD, RNA-binding domain; ED, effector domain; HSQC, heteronuclear single quantum coherence; TROSY, transverse relaxation optimized spectroscopy; SUMO, small ubiquitin-like modifier.

Contribution of NS1 Effector Domain Dimerization to Influenza A Virus Replication and Virulence

“…Here, we used the mouse-lethal A/Puerto Rico 8/34 (PR8) strain both to allow in vivo experiments and to negate the impact of mutation of W187 on the cellular pre-mRNA processing inhibition ability of NS1, which is inherently lacking in the NS1 of PR8 and many other strains (11,13,21). Consistent with previous studies implicating the NS1 ED helix-helix dimer in RBD function (1,12), poly(I·C) pulldown assays confirmed that the W187R mutation reduces dsRNA binding by NS1 (Fig. 1C).…”

“…As controls, wild-type (WT) NS1 bound efficiently to poly(I·C), while no binding was observed for the dsRNA-binding-incompetent R38A mutant (12,22). Although we noted that the effect of W187R on poly(I·C) binding was not as striking as that of a previously reported mutation (W187A) ((12; data not shown), our observation confirms that ED dimerization contributes to the overall affinity of full-length NS1 for dsRNA, which probably only occurs efficiently via cooperative binding of NS1 multimers (1,3).…”

“…The mechanisms underpinning NS1 multifunctionality are not fully understood, although multiple NS1 quaternary conformations promoted by weak, transient, homotypic interactions may provide one structural route to achieve and regulate such extensive functionality (12). Here, we extend earlier studies that focused on biochemical and structural characterization of the major homotypic conformer of the NS1 ED, namely, a weak helix-helix dimer mediated by the highly conserved tryptophan-187 (W187) (1,9,12,23,24) (Fig. 1A).…”

“…Future studies to dissect this caveat may therefore reveal a much greater biological impact of the highly conserved NS1 ED dimerization structural feature. Nevertheless, our present study provides the initial data to suggest biological relevance for the numerous structural and biophysical studies that have so far characterized NS1 ED helix-helix dimerization (1,9,12,23,24). …”

“…1B). This specific mutation in the A/Udorn/72 (Ud) NS1 has been shown to abrogate ED dimerization (1). Here, we used the mouse-lethal A/Puerto Rico 8/34 (PR8) strain both to allow in vivo experiments and to negate the impact of mutation of W187 on the cellular pre-mRNA processing inhibition ability of NS1, which is inherently lacking in the NS1 of PR8 and many other strains (11,13,21).…”