Viral infections, including those by single-stranded (ss) DNA viruses, positive (+)- and negative (-)-sense RNA viruses, produce immunostimulatory double-stranded RNA intermediates during viral replication cycle (reviewed by Chen YG & Hur S 2022). The presence of dsRNA is sensed by pattern recognition receptors (PRR) such as Toll-like receptor 3 (TLR3) which detects viral dsRNA in the lumen of the endosome (Alexopoulou L et al. 2001; Liu L et al. 2008; reviewed by Chen Y et al. 2021). Human respiratory syncytial virus (hRSV or RSV) induces TLR3 activity to mediate production of inflammatory cytokines and chemokines in RSV-infected human epithelial cells and fibroblasts (Rudd BD et al. 2005; Groskreutz DJ et al. 2006; Dou Y et al. 2013; Liu D et al. 2018). Increased TLR3 expression was detected in sputum samples from RSV-positive patients with acute exacerbations of chronic obstructive pulmonary disease (Liu D et al. 2018). In vivo studies in mice further confirm that TLR3 mediates host immune responses upon RSV infection (Rudd BD et al. 2006; Huang S et al. 2009; Aeffner F et al. 2011; Qi F et al. 2015).
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 and RIG-I-like receptors (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 TLR3. Upon ligand recognition, TLR3 recruits Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-β (TRIF or TICAM1) 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 (IFN-α, β).
