Characterization of wild-type and alternate transcription termination signals in the Rift Valley fever virus genome

Abstract

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a phlebovirus of the family Bunyaviridae, which affects humans and ruminants in Africa and the Middle East. RFV virus (RVFV) possesses a single-stranded tripartite RNA genome of negative/ambisense polarity. The S segment utilizes the ambisense strategy and codes for two proteins, the N nucleoprotein and the nonstructural NSs protein, in opposite orientations. The two open reading frames (ORFs) are separated by an intergenic region (IGR) highly conserved among strains and containing a motif, 5'-GCUGC-3', present on the genome and antigenome, which was shown previously to play a role in transcription termination (C. G. Albarino, B. H. Bird, and S. T. Nichol, J. Virol. 81:5246-5256, 2007; T. Ikegami, S. Won, C. J. Peters, and S. Makino, J. Virol. 81:8421-8438, 2007). Here, we created recombinant RVFVs with mutations or deletions in the IGR and showed that the substitution of the motif sequence by a series of five A's inactivated transcription termination at the wild-type site but allowed the transcriptase to recognize another site with the consensus sequence present in the opposite ORF. Similar situations were observed for mutants in which the motif was still present in the IGR but located close to the stop codon of the translated ORF, supporting a model in which transcription is coupled to translation and translocating ribosomes abrogate transcription termination. Our data also showed that the signal tolerated some sequence variations, since mutation into 5'-GCAGC-3' was functional, and 5'-GUAGC-3' is likely the signal for the termination of the 3' end of the L mRNA.

Fig. 1.

Mapping of the 3′ ends of RVFV N and NSs mRNAs of ZH₅₄₈. Shown are data for 3′ RACE analysis of the N and NSs mRNAs of ZH₅₄₈ expressed in cells collected at 6 h p.i. The sequences of the genome or antigenome template and the N or NSs mRNA sequences are aligned. The intergenic region (uppercase type) is identified from positions 1 to 82, and the motifs 5′-GCUGC-3′ and 5′-GCAGC-3′ are underlined in black and white, respectively. The N and NSs ORFs, including the stop codon, are represented by lowercase type. The arrows indicate the beginning of the poly(A) added in vitro.

Fig. 2.

The 5′-GCUGC-3′ motif in the IGR at positions 9 to 13 is responsible for N mRNA termination. (A) Plaques in Vero E6 cells formed by wild-type recombinant ZH₅₄₈ and mutated ZH-IGR-PA and ZH-IGR-PU. (B) Viral growth in Vero cells infected by wt and mutant ZH at an MOI of 2. (C) N and NSs mRNAs from ZH₅₄₈ and recombinant viruses mutated in the intergenic region of the S segment analyzed by Northern blotting. Total RNAs from Vero E6 cells uninfected or infected with recombinant viruses were harvested at 6 h p.i. and used for Northern blotting. The probes detected the N (left) or NSs (right) mRNA. NI, not infected; g, genome; ag, antigenome. (D and E) 3′ RACE analysis of the N (D) and NSs (E) mRNAs from Vero E6 cells infected with RVFV ZH-IGR-PA. The N and NSs ORFs are indicated by lowercase type, and the intergenic region is represented by uppercase type. The 3′ ends of the RNAs monitored by the beginning of the polyadenylation added in vitro are shown by arrows. The 5′-GCUGC-3′ motif and its complementary sequence are underlined in black and white, respectively. The mutations in the IGR are highlighted.

Fig. 3.

Rescue of recombinant ZH virus carrying a deleted IGR. (A) Schematic representation of the deletions in the S segment. NC, noncoding region. (B) Plaques in Vero E6 cells formed by wild-type recombinant ZH₅₄₈ and mutant strains ZH-IGR25 and ZH-IGR20. (C) Viral growth in Vero cells infected by wt and mutant ZH strains at an MOI of 2. (D) Expression of N and NSs mRNAs from the wild-type virus and the recombinant virus carrying the deleted intergenic region. Total RNA from Vero E6 cells uninfected or infected with recombinant viruses ZH-IGR25 to -IGR20 were harvested at 6 h p.i. and used for Northern blotting. The probes detected the N (top) or NSs (bottom) mRNA. (E and F) 3′ RACE analysis of the N (E) and NSs (F) mRNAs from Vero E6 cells infected with RVFV ZH-IGR25 (top) and ZH-IGR20 (bottom). The N and NSs ORFs are indicated by lowercase type, and the intergenic region is represented by uppercase type. The 3′ ends of the RNAs monitored by the beginning of the polyadenylation added in vitro are shown by arrows. The 5′-GCUGC-3′ motif and its complementary sequence are underlined in black and white, respectively.

Fig. 4.

Presence of alternate termination motifs in the S segment. (A) Plaques in Vero E6 cells formed by wild-type recombinant ZH₅₄₈ and the ZH-IGRpenta51-55 and -U:A11 mutants. (B) Viral growth in Vero cells infected by wt and mutant ZH strains at an MOI of 2. (C) N and NSs mRNAs from ZH₅₄₈ and recombinant viruses mutated in the intergenic region analyzed by Northern blotting. Total RNAs from Vero E6 cells uninfected or infected with recombinant viruses were harvested at 6 h p.i. and used for Northern blotting. The probes detected the N (left) or NSs (right) mRNA. (D and E) 3′ RACE analysis of the N (D) and NSs (E) mRNAs from Vero E6 cells infected with RVFV ZH-IGRpenta51-55 (top) and ZH-IGR-U:A11 (bottom). The N and NSs ORFs are indicated by lowercase type, and the intergenic region is represented by uppercase type. The 3′ ends of the RNAs monitored by the beginning of the polyadenylation added in vitro are shown by arrows. The 5′-GCUGC-3′ motif and its complementary sequence are underlined in black and white, respectively. The mutations are highlighted.

Fig. 5.

Mapping of the 3′ end of the L mRNA. (A) 3′ RACE analysis of L mRNA from ZH₅₄₈-infected cells collected at 10 h p.i. The 5′ noncoding sequence of the L segment is shown in the genomic sense from positions 1 to 90. The chromatogram of the RT-PCR product corresponding to the 3′ ends of the L antigenome/mRNA is presented. The initial position of the internal in vitro polyadenylation sequence is indicated by an arrow at position 28. (B) Sequence obtained from individual PCR products after cloning into pCRII-Topo plasmids. The sequences of the antigenome were obtained from 37 plasmids. The poly(A) added in vitro is not shown. The sequences highlighted in red indicate possible transcription termination motifs. (C) Alignment of the sequences surrounding the transcription termination site in the IGR of the genome (g), antigenome (ag), N, NSs, M, and L ORFs.

Fig. 6.

Schematic representation of the S segment of wild-type and mutated ZH viruses and their N and NSs mRNAs. The genome, antigenome, N, and NSs mRNAs of ZH₅₄₈ and mutant RVFVs are represented. The 5′ cap structure of the viral mRNAs is indicated. The 5′-GCUGC-3′ and 5′-GCAGC-3′ motifs are located on the genome and antigenome, shown by black and white boxes. The distance between the transcription termination signal and the codon stop is shown. The overlapping sequences at the 3′ ends of N and NSs mRNAs are also indicated.