Department of Medicine, University of Wisconsin-Madison School of Medicine and Public Health (SMPH), Madison, WI, United States.
Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, United States.
Front Immunol. 2023 Jul 26;14:1197356. doi: 10.3389/fimmu.2023.1197356. eCollection 2023.
The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood.
To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor.
We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer.
We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.
未折叠蛋白反应（UPR）已成为介导呼吸道合胞病毒（RSV）感染抗病毒防御的重要信号通路。早些时候，我们发现 RSV 复制主要激活进化上保守的肌醇需求酶 1α（IRE1α）-X 盒结合蛋白 1 剪接（XBP1s）分支的未折叠蛋白反应（UPR），产生炎症、代谢适应和细胞可塑性，但 UPR 增强炎症的机制尚不清楚。
为了更好地理解这一过程，我们通过 RNA-seq 和靶向切割和释放使用核酸酶（CUT&RUN）分析整合了基因组反应。这些数据与 RSV 感染的小气道细胞±IRE1α RNAse 抑制剂的 RNA-seq 分析相结合。
我们鉴定了 RSV 诱导的约 3200 个基因的表达变化；其中，279 个基因需要 IRE1α，并且富集在 IL-10/细胞因子信号通路中。从这个数据集，我们通过 CUT&RUN 确定了直接受 XBP1s 控制的基因。尽管 XBP1s 结合约 4200 个高可信度基因组结合位点，但只有一小部分 IL10/细胞因子信号基因直接结合。我们进一步应用 CUT&RUN 发现，RSV 感染增强了 XBP1s 在富含 AP1/Fra-1、RELA 和 SP1 结合位点的 786 个基因组位点上的加载。这些控制了一组细胞因子调节因子基因，包括 IFN 反应因子 1（IRF1）、CSF2、NFKB1A 和 DUSP10。关注 IRF1 的下游作用，选择性敲低（KD）和过表达实验表明，IRF1 的诱导控制 I 型和 III 型干扰素（IFN）和 IFN 刺激基因（ISG）的表达，表明 ISG 通过 XBP1 对 IRF1 的反式激活间接受到调控。在研究 IRF1 激活的机制时，我们观察到 XBP1s 直接结合 5'增强子序列，其 XBP1s 加载通过 RSV 增加。通过靶向 dCas9-KRAB 沉默子，减少 IRF1 激活，证明了增强子的功能要求。染色质免疫沉淀显示 XBP1 是 RSV 诱导的磷酸化 Ser2 Pol II 募集到增强子所必需的，但不是充分的。
我们得出结论，XBP1s 是 RSV 诱导的 IFN 和细胞因子调节基因核心亚组的直接激活剂。其中，IRF1 是 III 型 IFN 和 ISG 反应的上游。我们发现 RSV 调节 XBP1s 在 IRF1 5'增强子上的结合复合物，其激活是 IRF1 表达所必需的。这些发现为 IRE1α-XBP1s 通路增强气道黏膜抗病毒反应提供了新的见解。
