Coronavirus nucleocapsid protein facilitates template switching and is required for efficient transcription

Abstract

Purified nucleocapsid protein (N protein) from transmissible gastroenteritis virus (TGEV) enhanced hammerhead ribozyme self-cleavage and favored nucleic acid annealing, properties that define RNA chaperones, as previously reported. Several TGEV N-protein deletion mutants were expressed in Escherichia coli and purified, and their RNA binding ability and RNA chaperone activity were evaluated. The smallest N-protein domain analyzed with RNA chaperone activity, facilitating DNA and RNA annealing, contained the central unstructured region (amino acids 117 to 268). Interestingly, N protein and its deletion mutants with RNA chaperone activity enhanced template switching in a retrovirus-derived heterologous system, reinforcing the concept that TGEV N protein is an RNA chaperone that could be involved in template switching. This result is in agreement with the observation that in vivo, N protein is not necessary for TGEV replication, but it is required for efficient transcription.

FIG. 1.

Nucleic acid binding of N protein mutants. (A) Scheme of TGEV full-length N protein (wt, upper bar) and deletion mutants (ΔN, lower bars). Disorder-order pattern, residues corresponding to crystallized amino- and carboxy-terminal domains (NTD and CTD, respectively), and prediction of RNA binding residues according to the RNAbindR server (28, 29) are indicated. Labels on the left indicate the protein name. SR, Ser-rich domain; RRM, RNA recognition motif; NLS, nuclear localization signal; Ac, acidic domain. The numbers indicate the corresponding amino acids in N protein. (B) Western blot of protein fractions (upper panel) and Northwestern blot using different biotin-labeled probes (lower panels). Proteins were detected with an antibody specific for the GST tag (α-GST). Nondegraded protein species are indicated by black arrowheads. The nature of the nucleic acid probe is indicated at the right of the figure. The numbers on the left indicate molecular masses in kDa. (C) The intensity of the bands binding the probe was estimated by densitometry and corrected by the amount of intact protein in each case. Binding of N protein was considered to be 100 relative units (r.u.) of activity in each case; this threshold is indicated by the black dashed line. Error bars represent the standard deviations from four independent experiments.

FIG. 2.

Nucleic acid annealing assays. (A) DNA annealing assays were performed using an 18-mer biotin-labeled DNA oligonucleotide (Oligo C) and a 56-mer unlabeled DNA oligonucleotide (Oligo B) that under reaction conditions forms a stable secondary structure that must be unwound to allow double-stranded DNA formation. The ratio of dsDNA to ssDNA was estimated by densitometry (graph) and referenced to that obtained in the presence of wt N protein, considered 100 relative units (r.u.) (indicated by the black dashed line). (B) RNA annealing assays were performed using a biotin-labeled 16-mer RNA oligonucleotide representing the transcription regulating sequence of the leader (TRS-L) and an unlabeled 16-mer RNA oligonucleotide with the complementary sequence to the gene 7 TRS (cTRS-7). The ratio of dsRNA to ssRNA was estimated as for panel A. Error bars in panels A and B represent the standard deviations for results from four independent experiments.

FIG. 3.

In vitro template switching assay. (A) HIV-derived template switching system. Using a biotin-labeled DNA primer, HIV RT synthesizes a cDNA copy of a donor RNA of 131 nt (strong-stop DNA [SSDNA]). In the presence of an RNA chaperone, template switching occurs and a labeled transfer product (T) of 185 nt was obtained. R, repeated sequences, including transactivation response (TAR) element sequence. (B) Template switch assay in the presence of control NCp7 and GST proteins or TGEV N protein deletion mutants. (C) Template switch efficiency was estimated by densitometry of labeled T and SSDNA bands, and the transfer product amount was calculated as a percentage of the ratio T/(T + SSDNA) in each case. Error bars represent the standard deviations from four independent experiments.

FIG. 4.

In vivo coronavirus RNA synthesis. (A) Scheme of TGEV-derived replicons, containing full-length N protein (N wt) or lacking N protein (ΔN). A nonreplicative replicon (NR) containing full-length N protein, but unable to replicate due to a mutation affecting several replicase genes, was also used. All replicons contain gene 7 to allow measurement of transcription (1). (B) Quantification of negative and positive strands of genomic RNA (gRNA) using specific TaqMan assays. Experiments were performed as previously described (1), including a DNase I treatment to eliminate DNA from transfection. BHK-pSIN, BHK cells transfected with Sindbis virus replicon; BHK-pSIN-N, BHK cells expressing N protein from Sindbis virus replicon. (C) Transcription levels measured by quantification of subgenomic mRNA of gene 7, both negative and positive strand, using specific TaqMan assays. Error bars represent the standard deviations from five independent experiments.