MOE Joint International Research Laboratory of Animal Health and Food Safety, Engineering Laboratory of Animal Immunity of Jiangsu Province, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China.
Changjia (Shanghai) Agricultural Science and Technology Co., Ltd., Shanghai 201414, China.
Viruses. 2022 Jun 1;14(6):1206. doi: 10.3390/v14061206.
Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.
甲型流感病毒（IAV）调节宿主抗病毒反应以促进病毒生长和致病性。甲型流感病毒的非结构（NS1）蛋白在抑制宿主免疫反应方面发挥了不可或缺的作用，尤其是在限制干扰素（IFN）产生方面。在这项研究中，通过易错 PCR 向 A/WSN/1933（WSN，H1N1）的 NS1 基因中引入随机点突变，构建了随机突变质粒文库。通过反向遗传学生成了 NS1 随机突变病毒文库。为了筛选出未识别的 NS1 功能突变体，将文库病毒在小鼠中进行肺内传代，从第 4 代小鼠肺部中挑取单个蚀斑。Sanger 测序显示，从传代文库病毒中获得了 NS1 基因的 8 种不同突变。我们发现，NS1 F9Y 突变显著增强了病毒在体外（MDCK 和 A549 细胞）和体内（BALB/c 小鼠）的生长以及在小鼠中的毒力。NS1 D2I 突变削弱了病毒在体外和体内模型中的复制和致病性。进一步的研究表明，NS1 F9Y 突变病毒表现出系统性和选择性的细胞因子反应抑制作用，并抑制 IFN 的表达。此外，rNS1 D2I 病毒感染 A549 细胞后，先天免疫相关细胞因子的表达水平显著上调。总之，我们的结果表明，NS1 蛋白 N 端的两个突变可以改变 IAV 的病毒特性，并提供了额外的证据表明 NS1 蛋白是一个关键的毒力因子。这两个鉴定的 NS1 突变可能成为抗病毒药物和减毒疫苗开发的潜在靶点。
