State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Medical Research Institute, Wuhan University, Wuhan, China.
J Virol. 2018 May 14;92(11). doi: 10.1128/JVI.00149-18. Print 2018 Jun 1.
Nonstructural protein 1 (NS1) of influenza A virus regulates innate immune responses via various mechanisms. We previously showed that a naturally occurring deletion (the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus impairs the inhibition of type I interferon (IFN) in chicken fibroblasts and attenuates virulence in chickens. Here we found that the virus bearing this deletion in its NS1 effector domain showed diminished inhibition of IFN-related cytokine expression and attenuated virulence in mice. We further showed that deletion of the EALQR motif disrupted NS1 dimerization, impairing double-stranded RNA (dsRNA) sequestration and competitive binding with RIG-I. In addition, the EALQR-deleted NS1 protein could not bind to TRIM25, unlike full-length NS1, and was less able to block TRIM25 oligomerization and self-ubiquitination, further impairing the inhibition of TRIM25-mediated RIG-I ubiquitination compared to that with full-length NS1. Our data demonstrate that the EALQR deletion prevents NS1 from blocking RIG-I-mediated IFN induction via a novel mechanism to attenuate viral replication and virulence in mammalian cells and animals. H5 highly pathogenic avian influenza viruses have infected more than 800 individuals across 16 countries, with an overall case fatality rate of 53%. Among viral proteins, nonstructural protein 1 (NS1) of influenza virus is considered a key determinant for type I interferon (IFN) antagonism, pathogenicity, and host range. However, precisely how NS1 modulates virus-host interaction, facilitating virus survival, is not fully understood. Here we report that a naturally occurring deletion (of the EALQR motif) in the NS1 effector domain of an H5N1 swine-origin avian influenza virus disrupted NS1 dimerization, which diminished the blockade of IFN induction via the RIG-I signaling pathway, thereby impairing virus replication and virulence in the host. Our study demonstrates that the EALQR motif of NS1 regulates virus fitness to attain a virus-host compromise state in animals and identifies this critical motif as a potential target for the future development of small molecular drugs and attenuated vaccines.
甲型流感病毒的非结构蛋白 1（NS1）通过多种机制调节先天免疫反应。我们之前的研究表明，在 H5N1 猪源禽流感病毒的 NS1 效应结构域中存在一个天然缺失（EALQR 基序），这会削弱其对鸡成纤维细胞中 I 型干扰素（IFN）的抑制作用，并降低其在鸡中的毒力。在这里，我们发现该病毒在 NS1 效应结构域中存在此缺失时，其 IFN 相关细胞因子表达的抑制作用减弱，在小鼠中的毒力降低。我们进一步表明，EALQR 基序的缺失破坏了 NS1 二聚体的形成，削弱了双链 RNA（dsRNA）的隔离和与 RIG-I 的竞争性结合。此外，与全长 NS1 不同，缺失 EALQR 基序的 NS1 蛋白不能与 TRIM25 结合，并且更难以阻止 TRIM25 寡聚化和自身泛素化，与全长 NS1 相比，进一步削弱了对 TRIM25 介导的 RIG-I 泛素化的抑制作用。我们的数据表明，EALQR 缺失通过一种新机制阻止 NS1 阻断 RIG-I 介导的 IFN 诱导，从而减弱病毒在哺乳动物细胞和动物中的复制和毒力。H5 高致病性禽流感病毒已在 16 个国家感染了 800 多人，总病死率为 53%。在病毒蛋白中，流感病毒的非结构蛋白 1（NS1）被认为是 I 型干扰素（IFN）拮抗、致病性和宿主范围的关键决定因素。然而，NS1 如何调节病毒-宿主相互作用，促进病毒存活，尚不完全清楚。在这里，我们报告在 H5N1 猪源禽流感病毒的 NS1 效应结构域中存在一个天然缺失（EALQR 基序），破坏了 NS1 二聚体的形成，从而削弱了通过 RIG-I 信号通路阻断 IFN 诱导，从而损害了宿主中的病毒复制和毒力。我们的研究表明，NS1 的 EALQR 基序调节病毒适应性，以在动物中达到病毒-宿主妥协状态，并将该关键基序确定为未来开发小分子药物和减毒疫苗的潜在靶点。
