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.

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

“…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).…”

“…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).…”

“…NS1 acts at multiple levels to antagonize host interferon (IFN) responses, most notably by inhibiting the RIG-I/TRIM25 signaling axis (7), inhibiting posttranscriptional processing of host-cell mRNAs (including IFN-␤) (19), and directly limiting function of the dsRNA-activated antiviral effectors protein kinase R (PKR) and 2=-5=-oligo(A) synthetase (2=-5=-OAS) (2,14,18). 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.…”

“…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).…”

“…Indeed, rPR8 NS1-W187R virus induced slightly more IFN-␤ than rPR8 WT during infection. Our working model from structural data is that this NS1 ED helix-helix self-interaction is weak and transient (12), forming only to contribute to NS1 functions that require dsRNA binding, while dissociating for other NS1 functions, such as CPSF30 binding.…”