Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, 70101, Tainan, Taiwan.
Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, 70101, Tainan, Taiwan.
J Biomed Sci. 2021 Oct 5;28(1):66. doi: 10.1186/s12929-021-00764-0.
Influenza 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.
Luciferase 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.
Here 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.
Together 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.
甲型流感病毒（IAV）进化出各种策略来对抗宿主抗病毒防御以建立感染。甲型流感病毒（IAV）非结构蛋白 1（NS1）是一种关键的病毒因子，其主要通过靶向 RIG-I 信号来拮抗 I 型干扰素抗病毒反应。越来越多的证据表明，病毒 RNA 传感器 RIG-I、TLR3 和 TLR7 在不同的细胞类型中发挥作用，以检测 IAV RNA，从而诱导 I 型干扰素抗病毒反应来抵抗 IAV 感染。然而，目前尚不清楚 IAV NS1 是否可以利用一种共同的机制来同时拮抗这些 RNA 感应途径以产生 I 型干扰素，从而促进病毒在宿主中的传播。
荧光素酶报告基因检测用于确定 NS1 及其突变体对 RIG-I 和 TLR3 途径激活 IFN-β 和 NF-κB 启动子的影响。共免疫沉淀和共聚焦显微镜分析用于研究 NS1 和 TRAF3 之间的相互作用和共定位。泛素化测定用于研究 NS1 及其突变体对 TRAF3 泛素化的影响。通过反向遗传学生成携带 NS1 E152A/E153A 突变的重组突变病毒，用于生化、离体和体内分析，以探讨 NS1 E152/E153 残基在靶向 RNA 感应-TRAF3-I 型干扰素轴和 IAV 致病性中的重要性。
本研究报告 NS1 通过靶向 TRAF3 E3 泛素连接酶来颠覆 RIG-I、TLR3 和 TLR7 途径以产生 I 型干扰素。NS1 含有一个保守的 FTEE 基序（残基 150-153），其中 E152/E153 残基对于结合 TRAF3 以阻止这些 RNA 感应途径的 TRAF3 泛素化和 I 型干扰素产生至关重要。携带 NS1 E152A/E153A 突变的重组突变病毒在离体和体内诱导更高水平的 I 型干扰素产生，并在感染的小鼠中表现出减弱的表型，表明 E152/E153 残基在 IAV 致病性中的重要性。
本研究揭示了 IAV NS1 介导的免疫逃避的新机制，通过靶向 RNA 感应-TRAF3-I 型干扰素轴促进病毒感染。
