Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity

Chun-Yang Lin,Meng-Cen Shih,Kuan-Ru Chen,Kuan-Jung Lin,Jen-Ren Wang,Pin Ling,Ai-Li Shiau,Sheng-Wen Huang,Hung-Chun Chang,Lin-Fang Chen,Mei-Lin Yang,Sheng-Kai Ma

doi:10.1186/s12929-021-00764-0

Chun-Yang Lin, Meng-Cen Shih + Show 10 more

Open Access

https://doi.org/10.1186/s12929-021-00764-0

Copy DOI

Abstract

BackgroundInfluenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infection. The influenza A virus (IAV) non-structural protein 1 (NS1) is a key viral factor shown to counteract type I IFN antiviral response mainly through targeting RIG-I signaling. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. Yet, it remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, then promoting viral propagation in the host.MethodsLuciferase reporter assays were conducted to determine the effect of NS1 and its mutants on the RIG-I and TLR3 pathways to the activation of the IFN-β and NF-κB promoters. Coimmunoprecipitation and confocal microscopic analyses were used to the interaction and colocalization between NS1 and TRAF3. Ubiquitination assays were performed to study the effect of NS1 and its mutants on TRAF3 ubiquitination. A recombinant mutant virus carrying NS1 E152A/E153A mutations was generated by reverse genetics for biochemical, ex vivo, and in vivo analyses to explore the importance of NS1 E152/E153 residues in targeting the RNA sensing-TRAF3-type I IFN axis and IAV pathogenicity.ResultsHere we report that NS1 subverts the RIG-I, TLR3, and TLR7 pathways to type I IFN production through targeting TRAF3 E3 ubiquitin ligase. NS1 harbors a conserved FTEE motif (a.a. 150-153), in which the E152/E153 residues are critical for binding TRAF3 to block TRAF3 ubiquitination and type I IFN production by these RNA sensing pathways. A recombinant mutant virus carrying NS1 E152A/E153A mutations induces higher type I IFN production ex vivo and in vivo, and exhibits the attenuated phenotype in infected mice, indicating the importance of E152/E153 residues in IAV pathogenicity.ConclusionsTogether our work uncovers a novel mechanism of IAV NS1-mediated immune evasion to promote viral infection through targeting the RNA sensing-TRAF3-type I IFN axis.

Highlights

Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infec‐ tion
The non-structural protein 1 (NS1) effector domain counteracts RIG‐I signaling to type I IFN induction via an RNA binding‐independent manner First, we attempted to explore if IAV NS1 employs other mechanisms to subvert RIG-I signaling to type I IFN-mediated antiviral responses
Further experiments using IAV infected RNA indicated that NS1 and NS1RBD blocked RIG-I signaling to the IFN-β and NF-κB

Summary

Introduction

Influenza A virus (IAV) evolves strategies to counteract the host antiviral defense for establishing infec‐ tion. Growing evidence suggests that viral RNA sensors RIG-I, TLR3, and TLR7 function to detect IAV RNA in different cell types to induce type I IFN antiviral response to IAV infection. It remains unclear if IAV NS1 can exploit a common mechanism to counteract these RNA sensing pathways to type I IFN production at once, promoting viral propagation in the host. Type I IFNs act as a major driver to trigger antiviral innate immunity at the early infection via inducing hundreds of interferonstimulated genes (ISGs) that function to restrict viral propagation in host cells at the different steps of the viral life cycle [15, 38, 49]. Type I IFNs link to the induction of the adaptive immune responses that mediate a broad spectrum of antiviral immunity leading to viral clearance [38]

Methods

Results

Discussion

Conclusion