RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

The nucleocapsids (NCs) of the respiratory syncytial virus (RSV) can display multiple morphologies in vivo, including spherical, asymmetric, and filamentous conformations. Obtaining homogeneous ring-like oligomers in vitro is significant since they structurally represent one turn of the characteristic RSV NC helical filament. Here, we analyzed and optimized conditions for forming homogenous, recombinant nucleocapsid-like particles (NCLPs) of RSV in vitro. We examined the effects of modifying the integrated RNA length and sequence, altering incubation time, and varying buffer parameters, including salt concentration and pH, on ring-like NCLPs assembly using negative stain electron microscopy (EM) imaging. We showed that high-quality, homogeneous particles are assembled when incubating short, adenine-rich RNA sequences with RNA-free N associated with P (N⁰P). Further, we reported that a co-incubation duration greater than 3 days, a NaCl concentration between 100 mM and 200 mM, and a pH between 7 and 8 are optimal for N-RNA ring assembly with polyadenine RNA sequences. We believe assembling high-quality, homogeneous NCLPs in vitro will allow for further analysis of RSV RNA synthesis. This work may also lend insights into obtaining high-resolution nucleocapsid homogeneous structures for in vitro analysis of antiviral drug candidates against RSV and related viruses.