Abstract
The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N). Proper NC assembly is vital for RSV polymerase to engage the RNA template for RNA synthesis. Previous studies of NCs or nucleocapsid-like particles (NCLPs) from RSV and other nonsegmented negative-sense RNA viruses have provided insights into the overall NC architecture. However, in these studies, the RNAs were either random cellular RNAs or average viral genomic RNAs. An in-depth mechanistic understanding of NCs has been hampered by lack of an in vitro assay that can track NC or NCLP assembly. Here we established a protocol to obtain RNA-free N protein (N0) and successfully demonstrated the utility of a new assay for tracking assembly of N with RNA oligonucleotides into NCLPs. We discovered that the efficiency of the NCLP (N–RNA) assembly depends on the length and sequence of the RNA incorporated into NCLPs. This work provides a framework to generate purified N0 and incorporate it with RNA into NCLPs in a controllable manner. We anticipate that our assay for in vitro trackable assembly of RSV-specific nucleocapsids may enable in-depth mechanistic analyses of this process.
Highlights
The templates for transcription and replication by respiratory syncytial virus (RSV) polymerase are helical nucleocapsids (NCs), formed by viral RNAs that are encapsidated by the nucleoprotein (N)
The polymerase uses a single promoter in the 3Ј terminus of the genome and initiates and terminates mRNA transcription responding to gene start (GS) and gene end (GE) signals, respectively
Both length and sequence are critical for assembly of nucleocapsid-like particles (NCLPs), the longer length of RNA can compensate for the efforts of nonpreferred nucleotide-type RNA in NCLP assembly
Summary
B, schematic and cartoon view of the nucleoprotein. RBD, RNA-binding domain. PNTD is removed and assembled with the desired length and sequence of RNA to form N–RNA. Structures of the N0–P complex of human metapneumovirus, measles (MeV), Nipah virus, and vesicular stomatitis virus suggest a potential chaperone role of PNTD, preventing N from self-aggregating or binding to cellular RNA [11, 21, 22, 25,26,27]. We demonstrated the feasibility of large-scale preparation of N0 and in vitro assembly of trackable NCLPs. We used PNTD as a chaperone to prevent nonvirus-specific N–RNA interactions by coexpressing PNTD and N together, and we established a protocol to obtain large-scale soluble RSV N0–P complex. We showed that the purified N0–PNTD could be stimulated and assembled into NCLPs by adding RSV-specific RNA oligonucleotides and that PNTD is removed upon addition of RNA (Fig. 1D). This study established a powerful tool to further perform in-depth mechanistic studies of RSV RNA synthesis; in particular, cis-acting RNA signals and trans-acting viral proteins
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