Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Abstract

The interaction between the nucleocapsid (N) protein of mouse hepatitis virus (MHV) and RNA was studied in an effort to define portions of the N molecule that participate in binding to RNA. N mRNAs transcribed from SP6 and T7 vectors were translated in a rabbit reticulocyte lysate. Analysis of synthesized N protein in a nondenaturing gel system showed that it bound in vitro to an endogenous RNA in the reticulocyte lysate but not to its own mRNA. A set of deletion mutants was constructed in order to localize the RNA-binding activity of the N protein. It was found that removal of as much as 135 amino-terminal or 57 carboxy-terminal amino acids from the molecule had little or no effect on RNA binding. Moreover, deletion mutants lacking both termini still retained RNA-binding ability. By contrast, internal deletions or truncations extending beyond these two limits effectively abolished RNA binding by N protein. Thus, the RNA-binding region of N has been mapped to the second (central) of the three structural domains of the molecule.

Characterization of an Essential RNA Secondary Structure in the 3′ Untranslated Region of the Murine Coronavirus Genome

“…Mutations in these shorter synthetic donor RNAs were incorporated into the MHV genome by targeted recombination with the recipient virus Alb4 exactly as described previously (see Fig. 2B) (12,20,32). Negative results that were obtained with a subset of mutant donor RNAs by this method were then confirmed by attempting targeted recombination with donor RNAs transcribed from PacI-truncated vectors derived from pMH54.…”

“…Metabolic labeling of virus-specific RNA was carried out as previously described (12,32). In brief, L2 cells in spinner culture were infected with wild-type MHV at a multiplicity of 1 PFU per cell.…”

“…To facilitate the construction of mutations in the bulged stemloop in the 3Ј UTR, restriction sites adjacent to this structure were designed in pB36, the plasmid used to generate donor RNA for targeted recombination (12,32). Near the upstream boundary, we altered codons 450 and 451 of the N gene to create a unique MluI site ( Fig.…”

“…All plaque titrations and plaque purifications were performed with mouse L2 cells. Spinner cultures of L2 cells were maintained for RNA transfection by electroporation, as described previously (32). The interspecies chimeric coronavirus fMHV was grown in feline FCWF cells (22).…”

“…To enable more rapid construction of mutations in the 3Ј UTR, convenient restriction sites were incorporated into plasmid pB36, a T7 transcription vector that encodes a replicating MHV DI RNA comprising the 5Ј 467 nt of the MHV genome followed by a heterologous 48-nt linker, the entire N gene, the 3Ј UTR, and a 115-residue poly(A) tail (32). A unique MluI site was created 9 through 14 nt upstream of the N gene stop codon, and a unique EcoRV site was created 10 through 15 nt downstream of the bulged stem-loop structure (see Fig.…”