A transient homotypic interaction model for the influenza A virus NS1 protein effector domain.

Philip S Kerry,Benjamin G Hale,Claudia Hass,Margaret A Taylor,Andrew Lewis,Juan Ayllon,Adolfo García-Sastre,Rupert J Russell,Richard E Randall,Paul Digard

doi:10.1371/journal.pone.0017946

Philip S Kerry, Benjamin G Hale + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0017946

Copy DOI

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.

Highlights

During infection the influenza A virus NS1 protein participates in multiple protein-RNA and protein-protein interactions to perform a plethora of functions
We present several new crystal structure forms of different wild-type dimeric and rationally-designed monomeric NS1 effector domain (ED) grown under novel conditions, and confirm that the helix-helix ED dimer represents the major dimerization interface for this protein
Consistent with previous reports, both EDs were dimeric in solution as determined by gel filtration

Summary

Introduction

During infection the influenza A virus NS1 protein participates in multiple protein-RNA and protein-protein interactions to perform a plethora of functions (reviewed in [1]). 30 cellular and viral factors have been reported to interact either directly or indirectly with NS1, which seems surprising given that NS1 itself is relatively short (only ,230 amino-acids). Protein multifunctionality may be a key feature of many small RNA virus replication strategies given that they usually possess a restricted coding capacity. Potential mechanisms that likely influence the functions of NS1 include: (i) post-translational modifications [10,11,12]; (ii) strain-specific polymorphisms [13,14,15,16,17,18]; and (iii) spatio-temporal distribution [19,20]. As with many cellular proteins, different multimeric forms may be an important determinant of specific NS1 functions [21,22]

Methods

Results

Conclusion