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.

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.

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).

This Reactome event shows binding of RSV dsRNA species to RIG-I (DDX58).