title: A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.
abstract: Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins.
authors.affiliation: Biomedical Sciences Research Complex, University of St Andrews, St Andrews, Fife, United Kingdom.
authors.forename: Philip S
authors.initials: PS
authors.lastname: Kerry
authors.forename: Juan
authors.initials: J
authors.lastname: Ayllon
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Taylor
authors.forename: Claudia
authors.initials: C
authors.lastname: Hass
authors.forename: Andrew
authors.initials: A
authors.lastname: Lewis
authors.forename: Adolfo
authors.initials: A
authors.lastname: García-Sastre
authors.forename: Richard E
authors.initials: RE
authors.lastname: Randall
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Hale
authors.forename: Rupert J
authors.initials: RJ
authors.lastname: Russell
mesh_terms: D000818:Animals; D002460:Cell Line; D019295:Computational Biology; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004285:Dogs; D006801:Humans; D009980:Influenza A virus / Q000378:metabolism*; D016899:Interferon-beta / Q000235:genetics; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D018583:Pliability; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C085820:INS1 protein, influenza virus; D050505:Mutant Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D016899:Interferon-beta