Department of Disease Intervention and Prevention, Texas Biomedical Research Institute, San Antonio, Texas, USA.
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, Madrid, Spain.
J Virol. 2022 Mar 9;96(5):e0040821. doi: 10.1128/JVI.00408-21. Epub 2021 Apr 14.
PA-X is a nonstructural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacterium-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity compared to that of the wild-type A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacterium-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacterium-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. Highly pathogenic avian influenza viruses continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory nonstructural proteins, depending on the IAV type, subtype, or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. Here, we used, for the first time, a bacterium-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.
PA-X 是甲型流感病毒（IAV）的一种非结构蛋白，由聚合酶酸性（PA）N 端区域编码，该区域包含一个 C 端+1 移码序列。IAV PA-X 蛋白通过细胞 mRNA 切割，通过抑制宿主基因表达或细胞关闭来调节病毒诱导的宿主先天免疫反应和病毒致病性。高致病性禽流感病毒（HPAIV）的 H5N1 亚型自然感染不同的禽类物种，它们在家禽养殖中造成了巨大的经济影响，并且具有人畜共患和大流行的潜力，对人类公共卫生构成威胁。在本研究中，我们描述了一种基于细菌的新方法，用于鉴定 HPAIV A/Viet Nam/1203/2004 H5N1 的 PA-X 蛋白中对其抑制宿主蛋白表达或细胞关闭活性能力很重要的氨基酸残基。与野生型 A/Viet Nam/1203/2004 H5N1 PA-X 蛋白相比，鉴定的 PA-X 突变体显示出降低的关闭活性。值得注意的是，这种新的基于细菌的筛选方法使我们能够鉴定出广泛分布在 PA-X 整个 N 端区域的氨基酸残基。此外，我们发现一些影响 A/Viet Nam/1203/2004 H5N1 PA-X 宿主关闭活性的残基也会影响 minigenome 测定中的 PA 聚合酶活性。这些信息可用于设计针对 IAV 的新型更有效的抗病毒化合物。此外，我们的结果证明了使用这种基于细菌的方法来鉴定对抑制宿主基因表达的病毒蛋白活性很重要的氨基酸残基的可行性。高致病性禽流感病毒继续对全球动物和人类健康构成巨大威胁。尽管流感 A 病毒（IAV）的基因组大小有限，但该病毒根据 IAV 类型、亚型或菌株编码八个主要病毒结构蛋白和多个辅助非结构蛋白。IAV 的一种辅助蛋白 PA-X 是由聚合酶酸性（PA）蛋白编码的，通过调节 IAV 诱导的宿主炎症和先天免疫反应参与致病性。然而，IAV PA-X 调节宿主免疫反应的分子机制尚不清楚。在这里，我们首次使用基于细菌的方法鉴定对 IAV PA-X 诱导宿主关闭活性能力很重要的氨基酸，并描述了与抑制宿主基因表达能力相关的新型残基及其在 PA 聚合酶活性中的贡献。
