**5.2 Transcription and replication**

RSV replication and transcription are dependent on viral components including viral RNA, N, P, L, and M2–1 [131]. RSV utilizes its own machinery (RNP complex) to replicate in the host cytoplasm [132]. Inclusion body formation is a hallmark of RSV infection produced by multiple viral proteins including N, P, L, and M2–1 and this cytoplasmic structure is increased with RSV infection in epithelial cells [72, 133, 134]. Specifically, N and P proteins are important for inclusion body formation because the expression of these proteins with or without RSV infection showed inclusion body formation [135]. P protein can hijack host cell machinery by forming a complex with host phosphatase (PP1) and this P-PP1 complex dephosphorylates M2–1, as a result, P protein can recruit M2–1 protein in the inclusion body to facilitate viral RNA synthesis [136]. M protein is also reported to localize in inclusion bodies mediated by M2–1 protein [137]. The inclusion body is thought to be the first place where M protein interacts with the ribonucleoprotein complex and M protein is

involved in the release of RNP from inclusion bodies towards budding [138]. Host actin cytoskeleton and Hsp70 proteins are also observed in inclusion bodies, but their role is not clear yet and they perhaps facilitate viral machinery [139]. RSV infection causes vigorous stress on the host cell resulting formation of cytoplasmic stress granules, which are different from cytoplasmic inclusion bodies and these stress granule formations facilitates viral replication [140].

Both viral RNA replication and mRNA transcription start from the same single promoter in leader (le) region (44-nucleotide long) at the 3′ end of RSV genome and it produces methyl-guanosine capped and polyadenylated mRNA during transcription and antigenome during replication [20, 141–143]. Each RSV gene has two conserved cis-acting elements including a gene start (gs) signal at the beginning and a gene end (ge) signal at the end [144]. The promoter of leader (Le) region at the 3′ end of RSV genome has two initiation sites, one is at position +1 or 1 U required for replication and another one is at position +3 or 3C required for transcription [145]. 9 out of 10 gs signaling sequences are highly conserved whereas the tenth one has minimal sequence difference in RSV genome [19]. During transcription, both gs and ge signaling sequences play significant role, specifically, gs signal provides direction to RNA-dependent RNA-polymerase (RdRp) for initiating RNA synthesis and ge signal provides direction to RdRp to polyadenylate and release the mRNA [146, 147]. Then RdRp connected to the template can initiate transcription again at the next gs signal and this process persists along RSV genome [144]. During replication, RdRp attaches a similar promoter sequence in le region, but it ignores ge signal and continues to proceed throughout the genome to produce an antigenome, which is a full-length positive-sense complement of RSV genome [145]. Viral genome and antigenome RNA are encapsidated in RSV nucleoprotein whereas viral mRNAs are not encapsidated [145]. Every nucleoprotein monomer interacts with 7 nucleotides of viral RNA and this complex forms a helical nucleocapsid acting as a template for the next RNA synthesis. This encapsidation is thought to increase RdRp activities to override ge signal during replication, therefore, encapsidation is the distinguishing factor between replication and transcription [23, 148, 149]. The trailer (tr) region (155-neucleotide long) at the 3′ end of RSV antigenome has a promoter, which allows RdRp towards RSV genome synthesis [142, 143, 150]. The first 12 nucleotides of tr promoter are like those of the le promoter and the signal starts from position +1 and + 3 undergoes replication and transcription, respectively, but tr promoter cannot produce capped and polyadenylated mRNA because of lacking ge signal sequence adjacent to tr promoter [151, 152]. However, it is reported that tr promoter can initiate transcription of short RNA, which can inhibit cellular stress granules [153]. The concentration of ATP or GTP can determine the fate of replication and transcription at positions +1 (1 U) or position +3 (3C) observed at in vitro model, specifically, higher ATP concentration stimulates initiation from 1 U and evades initiation at 3C, in contrast, higher GTP concentration displays opposite effect [154]. Overall, L and P proteins form the core RdRp and L-P complex then form L-P-N and L-P-M2–1 complex to initiate replication and transcription, respectively [79, 155].
