School of Public Health, National Defense Medical Center, Taipei, Taiwan.
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan.
Front Cell Infect Microbiol. 2022 Sep 15;12:998584. doi: 10.3389/fcimb.2022.998584. eCollection 2022.
Non-structural protein 1 (NS1), one of the viral proteins of influenza A viruses (IAVs), plays a crucial role in evading host antiviral immune response. It is known that the IAV NS1 protein regulates the antiviral genes response mainly through several different molecular mechanisms in cytoplasm. Current evidence suggests that NS1 represses the transcription of gene by inhibiting the recruitment of Pol II to its exons and promoters in infected cells. However, IAV NS1 whether can utilize a common mechanism to antagonize antiviral response by interacting with cellular DNA and immune-related transcription factors in the nucleus, is not yet clear.
Chromatin immunoprecipitation and sequencing (ChIP-seq) was used to determine genome-wide transcriptional DNA-binding sites for NS1 and NF-κB in viral infection. Next, we used ChIP-reChIP, luciferase reporter assay and secreted embryonic alkaline phosphatase (SEAP) assay to provide information on the dynamic binding of NS1 and NF-κB to chromatin. RNA sequencing (RNA-seq) transcriptomic analyses were used to explore the critical role of NS1 and NF-κB in IAV infection as well as the detailed processes governing host antiviral response.
Herein, NS1 was found to co-localize with NF-κB using ChIP-seq. ChIP-reChIP and luciferase reporter assay confirmed the co-localization of NS1 and NF-κB at type III IFN genes, such as , , and . We discovered that NS1 disturbed binding manners of NF-κB to inhibit expression. NS1 hijacked NF-κB from a typical promoter to the exon-intron region of and decreased the enrichment of RNA polymerase II and H3K27ac, a chromatin accessibility marker, in the promoter region of during IAV infection, consequently reducing gene expression. NS1 deletion enhanced the enrichment of RNA polymerase II at the promoter and promoted its expression.
Overall, NS1 hijacked NF-κB to prevent its interaction with the promoter and restricted the open chromatin architecture of the promoter, thereby abating antiviral gene expression.
甲型流感病毒（IAV）的非结构蛋白 1（NS1）是病毒蛋白之一，在逃避宿主抗病毒免疫反应方面发挥着关键作用。已知 IAV NS1 蛋白主要通过几种不同的分子机制在细胞质中调节抗病毒基因的反应。目前的证据表明，NS1 通过抑制 Pol II 与感染细胞中外显子和启动子的募集来抑制基因的转录。然而，IAV NS1 是否可以通过与细胞核中的细胞 DNA 和免疫相关转录因子相互作用利用共同的机制来拮抗抗病毒反应，尚不清楚。
使用染色质免疫沉淀和测序（ChIP-seq）来确定 NS1 和 NF-κB 在病毒感染中的全基因组转录 DNA 结合位点。接下来，我们使用 ChIP-reChIP、荧光素酶报告基因测定和分泌型碱性磷酸酶（SEAP）测定来提供有关 NS1 和 NF-κB 动态结合染色质的信息。RNA 测序（RNA-seq）转录组分析用于探索 NS1 和 NF-κB 在 IAV 感染中的关键作用以及控制宿主抗病毒反应的详细过程。
在此，通过 ChIP-seq 发现 NS1 与 NF-κB 共定位。ChIP-reChIP 和荧光素酶报告基因测定证实了 NS1 和 NF-κB 在 III 型 IFN 基因（如 、 和 ）上的共定位。我们发现 NS1 扰乱了 NF-κB 的结合方式以抑制 表达。NS1 将 NF-κB 从典型的 启动子劫持到 和 的外显子-内含子区域，并在 IAV 感染过程中降低 RNA 聚合酶 II 和 H3K27ac（一种染色质可及性标记）在 启动子区域的富集，从而降低 基因表达。NS1 缺失增强了 RNA 聚合酶 II 在 启动子上的富集并促进了其表达。
总体而言，NS1 劫持了 NF-κB，以防止其与 启动子相互作用，并限制了启动子的开放染色质结构，从而减弱了抗病毒基因的表达。
