RSV dsRNA binds DDX58

Retinoic acid-inducible gene I (RIG-I)-like receptors (RIG-I and MDA5), encoded by the DDX58 and IFIH1 genes, respectively, detect the presence of double-stranded RNA within the cell cytosol (reviewed by Chow KT et al. 2018; Rehwinkel J & Gack MU 2020). DDX58 (RIG-I) is activated by short 5' triphosphorylated dsRNA, while IFIH1 (MDA5) is triggered by long double-stranded RNA molecules regardless of their 5' phosphorylation status. Upon detection, both RIG-I and MDA5 transmit signals through the adaptor protein mitochondrial antiviral-signaling protein (MAVS) to induce the activation of transcription factors interferon receptor factor 3 (IRF-3) and nuclear factor kappa B (NF-kappa-B), which ultimately results in the induction of type I interferons.<p>Respiratory syncytial virus (RSV) induces DDX58 (RIG-I) activity to mediate production of inflammatory cytokines and chemokines (Liu P et al. 2007; Okabayashi T et al. 2011; Guo X et al. 2015; Martín-Vicente M et al. 2019). Immunoprecipitation assay showed that DDX58 (RIG-I) binds to RSV RNA (Liu P et al. 2007). Upregulation of DDX58 expression was observed following RSV infection in human bronchiolar carcinoma cell line A549 (Liu P et al. 2007; Guo X et al. 2015) and in human primary peripheral blood mononuclear cells (PBMCs) (Vissers M et al. 2012). Both type I IFN-α/β and type III IFN-λ were induced by RSV infection in A549 cells in a DDX58 (RIG-I)-dependent manner (Okabayashi T et al. 2011). Suppression of DDX58 in RSV-infected human primary nasal epithelial cells (hTERT-NECs) reduced the production of IFN-λ, but not type I IFNs, indicating that DDX58 (RIG-I) plays a prominent role in inducing type III IFN in NECs (Okabayashi T et al. 2011). RSV-induced phosphorylation of NF-kappa B p65 at serine 536 in A549 cells was shown to be dependent upon the presence of RIG-I, MAVS, TRAF6, and IKK beta (Yoboua F et al. 2010). Silencing DDX58(RIG-I) in A549 cells using siRNA inhibits the activation of both NF-κB and IRF3 transcription factors, as well as the expression of IFN-β, CXCL10, CCL5, ISG15, TNF-α, and IL-6, during early stages of RSV infection (Liu P et al. 2007; Yamamoto K et al. 2016; Martín-Vicente M et al. 2019). Additionally, siRNA-mediated suppression of RIG-I expression in A549 or Vero cells blocks DDX58 (RIG-I) induction by RSV infection, preventing the upregulation of NLRC5, MHC-I, and the phosphorylation of IRF3, all of which are essential for an effective IFN response (Guo X et al. 2015). Further, immunofluorescence assay showed that DDX58 (RIG-I), IFIH1 (MDA5) and MAVS co-localized with RSV genomic RNA (RSV A2 strain) in the small inclusion bodies in A549 cells (Lifland AW et al. 2012). Pretreatment of human, mouse, or ferret airway cell lines with a synthetic DDX58 (RIG-I) agonist (3pRNA) before RSV infection reduces susceptibility to the virus and inhibits its replication (Schwab LSU et al. 2022). A single intravenous injection of mice or ferrets with 3pRNA prior to RSV infection significantly restricts virus growth in the lungs (Schwab LSU et al. 2022). Clinical studies have shown a positive correlation between RSV viral load and the expression of RIG-1 mRNA in infants with RSV-induced bronchiolitis (Scagnolari C et al. 2009). The significance of the DDX58-signaling pathway was also demonstrated in an in vivo infectious model using MAVS-deficient mice, which exhibited reduced IFN-I production and pro-inflammatory cytokine expression in response to RSV (Demoor T et al. 2012; Kirsebom FCM et al. 2019; Paulsen M et al. 2020). These findings suggest a crucial role for DDX58 (RIG-I) in host defense against RSV infection.<p>The replication/transcription process of RSV, a (−)-sense ssRNA virus, is coordinated by nucleoprotein (N), large protein (L), phosphoprotein (P), and M2-1 protein. Viral N, L, P , M2-1 and RSV RNA form the RNA synthesis ribonucleoprotein (RNP) complex (reviewed by Cao D et al. 2021). This complex serves as a template for RNA replication, generating the (+) RNA antigenome and the (-) RNA genome, as well as for RNA transcription, producing capped and poly-adenylated mRNAs. Within this complex, the catalytic core L polypeptide performs various enzymatic activities including RNA-dependent RNA polymerase (RdRp) activity, polyribonucleotidyl transferase activity, which is essential for mRNA 5' cap addition, and methyltransferase activity to catalyze the cap methylation at both N7- and 2′-O-positions (reviewed by Sutto-Ortiz P et al. 2023). The interaction of the cofactor P with multiple proteins, including L and M2-1, enables conformational changes necessary to perform various enzymatic activities during the viral replication/transcription process. The M2-1 protein is required for RSV transcription to prevent premature transcription termination by increasing the processivity of the RdRp complex. Further, during RNA replication, the viral RNA polymerase activity can generate both standard (such as genomic) viral RNA and nonstandard viral RNA (reviewed by Vignuzzi M  & López CB 2019; González Aparicio LJ et al. 2022). One type of nonstandard viral genome is known as copy-back viral genomes (cbVGs). These cbVGs are produced when the polymerase enzyme dissociates from the template strand at a specific breakpoint and then resumes elongation at a downstream rejoin point (reviewed by Vignuzzi M  & López CB 2019; González Aparicio LJ et al. 2022). This process creates a complementary end to the 5' end of the nascent genomic RNA, resulting in the formation of double-stranded cbVG structures. RSV infection has been shown to generate cbVGs in vitro and in vivo (Sun Y et al. 2015; 2019; Felt SA et al. 2022). The accumulation of cbVGs is thought to modulate the viral replication and stimulate host immune responses via PRRs including TLR3, DDX58 and IFIH1 (reviewed by Vignuzzi M  & López CB 2019; González Aparicio LJ et al. 2022).<p>This Reactome event shows binding of RSV dsRNA species to RIG-I (DDX58).

维甲酸诱导基因I（RIG-I）样受体（RIG-I和MDA5），分别由DDX58和IFIH1基因编码，可在细胞质内检测到双链RNA的存在（参见Chow KT等，2018年；Rehwinkel J & Gack MU，2020年综述）。DDX58（RIG-I）被短5'三磷酸化双链RNA激活，而IFIH1（MDA5）则由长双链RNA分子触发，不受其5'磷酸化状态的影响。在检测到后，RIG-I和MDA5通过适配蛋白线粒体抗病毒信号蛋白（MAVS）传递信号，诱导转录因子干扰素受体因子3（IRF-3）和核因子κB（NF-κB）的激活，最终导致I型干扰素的诱导。<p>呼吸道合胞病毒（RSV）通过诱导DDX58（RIG-I）活性，介导炎症细胞因子和趋化因子的产生（参见Liu P等，2007年；Okabayashi T等，2011年；Guo X等，2015年；Martín-Vicente M等，2019年）。免疫沉淀实验表明DDX58（RIG-I）与RSV RNA结合（参见Liu P等，2007年）。在人类细支气管癌细胞系A549（参见Liu P等，2007年；Guo X等，2015年）和人类原发性外周血单个核细胞（PBMCs）（参见Vissers M等，2012年）中观察到DDX58表达的上调。在A549细胞中，RSV感染诱导I型IFN-α/β和III型IFN-λ的产生，均依赖于DDX58（RIG-I）（参见Okabayashi T等，2011年）。在RSV感染的人原代鼻上皮细胞（hTERT-NECs）中抑制DDX58（RIG-I）的表达，会减少IFN-λ的产生，但不会影响I型干扰素，这表明DDX58（RIG-I）在NECs中诱导III型干扰素的过程中发挥着显著作用（参见Okabayashi T等，2011年）。在A549细胞中，RSV诱导的NF-kappa B p65在丝氨酸536位的磷酸化被发现依赖于RIG-I、MAVS、TRAF6和IKK beta的存在（参见Yoboua F等，2010年）。使用siRNA沉默A549细胞中的DDX58（RIG-I），抑制了NF-κB和IRF3转录因子的激活，以及IFN-β、CXCL10、CCL5、ISG15、TNF-α和IL-6的表达，这些都是在RSV感染早期阶段发生的（参见Liu P等，2007年；Yamamoto K等，2016年；Martín-Vicente M等，2019年）。此外，通过siRNA抑制A549或Vero细胞中RIG-I的表达，可以阻断RSV感染诱导的DDX58（RIG-I）的诱导，防止NLRC5、MHC-I的上调和IRF3的磷酸化，所有这些对于有效的IFN反应都是必不可少的（参见Guo X等，2015年）。进一步的免疫荧光实验表明，DDX58（RIG-I）、IFIH1（MDA5）和MAVS在A549细胞的小包涵体中与RSV基因组RNA（RSV A2株）共定位（参见Lifland AW等，2012年）。在RSV感染前，预先用合成的DDX58（RIG-I）激动剂（3pRNA）处理人、小鼠或雪貂的气道细胞系，可以降低病毒易感性并抑制其复制（参见Schwab LSU等，2022年）。在RSV感染前，给小鼠或雪貂静脉注射3pRNA可以显著限制肺部病毒的生长（参见Schwab LSU等，2022年）。临床研究表明，RSV病毒载量与RSV引起的细支气管炎婴儿中RIG-1 mRNA的表达之间存在正相关（参见Scagnolari C等，2009年）。在MAVS缺陷型小鼠体内感染模型中，DDX58-信号通路的重要性也得到了证实，这些小鼠对RSV的免疫反应表现为IFN-I的产生减少和促炎细胞因子表达降低（参见Demoor T等，2012年；Kirsebom FCM等，2019年；Paulsen M等，2020年）。这些发现表明DDX58（RIG-I）在宿主防御RSV感染中起着至关重要的作用。<p>RSV（-）感单链RNA病毒的复制/转录过程由核蛋白（N）、大蛋白（L）、磷酸蛋白（P）和M2-1蛋白协调。Viral N、L、P、M2-1和RSV RNA形成RNA合成核糖核蛋白（RNP）复合物（参见Cao D等，2021年综述）。该复合物作为RNA复制的模板，生成（+）RNA抗原组和（-）RNA基因组，以及RNA转录的模板，产生加帽和聚腺苷酸化的mRNA。在此复合物中，催化核心L多肽执行各种酶促活性，包括RNA依赖性RNA聚合酶（RdRp）活性、聚核糖核苷酸转移酶活性，这对于mRNA 5'帽的添加至关重要，以及甲基转移酶活性，催化N7-和2′-O-位置的帽甲基化（参见Sutto-Ortiz P等，2023年综述）。辅助因子P与包括L和M2-1在内的多种蛋白质相互作用，使病毒复制/转录过程中进行各种酶促活性的构象变化成为可能。M2-1蛋白对于RSV转录至关重要，它通过增加RdRp复合物的进程性，防止过早的转录终止。此外，在RNA复制过程中，病毒RNA聚合酶活性可以生成标准的（如基因组）病毒RNA和非标准的病毒RNA（参见Vignuzzi M & López CB，2019年；González Aparicio LJ等，2022年综述）。一种非标准病毒基因组称为回文病毒基因组（cbVGs）。当聚合酶酶在特定的断点从模板链解离，然后在下游的重新连接点重新延长时，会产生cbVGs。这个过程在5'端新生基因组RNA上创建一个互补端，导致形成双链cbVG结构。体外和体内研究表明，RSV感染可以生成cbVGs（参见Sun Y等，2015年；2019年；Felt SA等，2022年）。cbVGs的积累被认为可以通过PRRs（如TLR3、DDX58和IFIH1）调节病毒复制并刺激宿主免疫反应（参见Vignuzzi M & López CB，2019年；González Aparicio LJ等，2022年综述）。<p>本Reactome事件显示了RSV双链RNA与RIG-I（DDX58）的结合。