ABSTRACT

Highly pathogenic avian influenza viruses (HPAIVs) frequently receive global attention as threats to public health. The NS1 protein is a key virulence factor known to impair host antiviral responses. The study herein revealed HPAIV H5N2 NS gene encoded additional protein; a truncated NS1 variant, designated NS3, produced by alternative splicing of the NS transcript. To examine the function of NS3 during infection, we generated recombinant viruses expressing either full-length NS1 (RG-AIV-T375G) or NS3 (RG-AIV-NS3). Interestingly, RG-AIV-NS3 virus produced higher titres than RG-AIV-T375G in multiple mammalian cell lines. However, RG-AIV-T375G exhibited a replication advantage over RG-AIV-NS3 in chicken DF-1 cells, indicating that host cell identity dictates the effect of NS3 on viral replication. In mice and mammalian cells, RG-AIV-NS3 infection elicited higher level of cytokines, including IFN-β, MX and TNF-α, potentially due to its higher replication activity. Based on mini-genome assay, NS3 had pronounced effects on viral replication machinery. Surprisingly, NS3 retained an interaction with PKR and suppressed PKR activation despite its lack of amino-acid residues 126-167. The poor replication ability of RG-AIV-T375G was partially restored in cells deficient in PKR suggesting that full-length NS1 may be insufficient to suppress PKR function. Notably, virulence of the full-length NS1-expressing RG-AIV-T375G virus was highly attenuated in mice when compared to RG-AIV-NS3. In summary, our study reveals the existence and function of a previously unidentified H5N2 viral protein, NS3. We found that NS3 is functionally distinct from NS1 protein, as it enhances viral replication and pathogenicity in mammalian systems, potentially via suppression of PKR activity.

摘要

高致病性禽流感病毒 (HPAIV) 经常作为对公共卫生的威胁而受到全球关注。NS1 蛋白是已知会损害宿主抗病毒反应的关键毒力因子。本文的研究揭示了 HPAIV H5N2 NS 基因编码额外的蛋白质；一种截短的 NS1 变体，称为 NS3，由 NS 转录本的可变剪接产生。为了检查 NS3 在感染期间的功能，我们生成了表达全长 NS1 (RG-AIV-T375G) 或 NS3 (RG-AIV-NS3) 的重组病毒。有趣的是，RG-AIV-NS3 病毒在多种哺乳动物细胞系中产生的滴度高于 RG-AIV-T375G。然而，RG-AIV-T375G 在鸡 DF-1 细胞中表现出优于 RG-AIV-NS3 的复制优势，表明宿主细胞身份决定了 NS3 对病毒复制的影响。在小鼠和哺乳动物细胞中，RG-AIV-NS3 感染引起更高水平的细胞因子，包括 IFN-β、MX 和 TNF-α，可能是由于其更高的复制活性。基于微型基因组测定，NS3对病毒复制机制有显着影响。令人惊讶的是，NS3 保留了与 PKR 的相互作用并抑制了 PKR 的激活，尽管它缺乏 126-167 位氨基酸残基。RG-AIV-T375G 较差的复制能力在缺乏 PKR 的细胞中得到部分恢复，这表明全长 NS1 可能不足以抑制 PKR 功能。值得注意的是，与 RG-AIV-NS3 相比，表达全长 NS1 的 RG-AIV-T375G 病毒在小鼠中的毒力高度减弱。总之，我们的研究揭示了以前未鉴定的 H5N2 病毒蛋白 NS3 的存在和功能。我们发现 NS3 在功能上不同于 NS1 蛋白，