A double-stranded RNA platform is required for the interaction between a host restriction factor and the NS1 protein of influenza A virus.

Guifang Chen,Robert M Krug,Gaetano T Montelione,Li-Chung Ma,Ryan L Woltz,Emily M Grasso,Shanshan Wang

doi:10.1093/nar/gkz1094

Guifang Chen, Robert M Krug + Show 5 more

Open Access

https://doi.org/10.1093/nar/gkz1094

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Abstract

Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.

Highlights

Influenza A viruses cause an annual contagious respiratory human disease, and are responsible for periodic pandemics that result in high mortality [1]
We show that the dsRNAbinding activities of both DHX30 and the NS1 protein are required for the interaction of these two proteins, and that both double-stranded RNA (dsRNA)-binding activities are required because a dsRNA platform that binds both NS1 and DHX30 mediates the interaction between these two proteins
Previous studies have demonstrated that the binding of several different proteins to the influenza A virus NS1 protein requires the same two amino-acid residues, Arg38 and Lys41, in the NS1 RNA-binding domain (RBD) that are required for dsRNA binding [6,7,8,9,10,11,12]

Summary

Introduction

Influenza A viruses cause an annual contagious respiratory human disease, and are responsible for periodic pandemics that result in high mortality [1]. The smallest segment encodes the NS1 protein, a small nonstructural protein that plays many crucial roles in virus infection, including inhibiting host antiviral responses, regulating other cellular and viral functions, and impacting virulence and virus-induced pathogenesis [3,4]. One cellular protein associated with these complexes is the DDX21 RNA helicase, a host restriction factor that binds the PB1 viral polymerase subunit, thereby suppressing viral RNA synthesis and viral protein synthesis at early times after infection [6]. DDX21mediated antiviral activity is countered by the NS1 protein, which binds DDX21 and displaces PB1 from DDX21. These results prompted us to examine the potential antiviral activities of other cellular proteins in NS1–CPSF30 com-

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