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. 1999 Oct 12;96(21):12056-61.
doi: 10.1073/pnas.96.21.12056.

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

G van Marle  1 , J C Dobbe, A P Gultyaev, W Luytjes, W J Spaan, E J Snijder
Affiliations

Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences

G van Marle et al. Proc Natl Acad Sci U S A. .

Abstract

To generate an extensive set of subgenomic (sg) mRNAs, nidoviruses (arteriviruses and coronaviruses) use a mechanism of discontinuous transcription. During this process, mRNAs are generated that represent the genomic 5' sequence, the so-called leader RNA, fused at specific positions to different 3' regions of the genome. The fusion of the leader to the mRNA bodies occurs at a short, conserved sequence element, the transcription-regulating sequence (TRS), which precedes every transcription unit in the genome and is also present at the 3' end of the leader sequence. Here, we have used site-directed mutagenesis of the infectious cDNA clone of the arterivirus equine arteritis virus to show that sg mRNA synthesis requires a base-pairing interaction between the leader TRS and the complement of a body TRS in the viral negative strand. Mutagenesis of the body TRS of equine arteritis virus RNA7 reduced sg RNA7 transcription severely or abolished it completely. Mutations in the leader TRS dramatically influenced the synthesis of all sg mRNAs. The construction of double mutants in which a mutant leader TRS was combined with the corresponding mutant RNA7 body TRS resulted in the specific restoration of mRNA7 synthesis. The analysis of the mRNA leader-body junctions of a number of mutants with partial transcriptional activity provided support for a mechanism of discontinuous minus-strand transcription that resembles similarity-assisted, copy-choice RNA recombination.

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Figures

Figure 1
Figure 1
Schematic diagram of the EAV genome organization and expression. The regions of the genome specifying the leader (L) sequence, the two large replicase ORFs (ORFs 1a and 1b), and the structural genes are indicated. The nested set of seven EAV mRNAs (genome and sg mRNAs 2–6) is depicted below. The black boxes in the genomic RNA indicate the position of leader and body TRSs.
Figure 2
Figure 2
Immunofluorescence analysis of BHK-21 cells transfected with wild-type EAV RNA or with mutants L3, B3, or LB3. (a) At 12 h posttransfection, cells were fixed and double-stained for nsp2 (Upper) and N (Lower) to test for genome replication and mRNA7 synthesis, respectively. (b) Double labeling for nsp2 (Upper) and GL (Lower) to monitor genome replication and mRNA5 transcription, respectively.
Figure 3
Figure 3
Analysis of genome and sg mRNA synthesis by leader and body TRS mutants. At 12 h posttransfection, total i.c. RNA was isolated, separated in denaturing 1.5% agarose gels, and hybridized (18) to a radiolabeled oligonucleotide probe complementary to the 3′ end of the genome. (a) Analysis of constructs containing single or double C-to-G substitutions in leader TRS (L series), RNA7 body TRS (B series), or both TRSs (LB series). (b) Analysis of a similar set of mutants in which the entire TRS has been mutated (5′-UCAAC-3′ to 5′-AGUUG-3′).
Figure 4
Figure 4
Sequence analysis of mRNA7 leader–body junction sequences. Total i.c. RNA was isolated from transfected cells and used for an mRNA7-specific RT-PCR. The leader–body junction sequences of sg mRNA7 were determined by direct sequence analysis of the RT-PCR products (see also Table 1). The position of the TRS at the mRNA7 leader–body junction is indicated, as are reference nucleotides from the leader region (genome position C-192) and the RNA7 body (U-12268). (a) Analysis of the leader–body junction sequence of the sg mRNA7 produced by the wild-type construct and by mutant LB1, containing both mutant leader and RNA7 body TRSs (5′-UGAAC-3′). (b) Sequence analysis of the leader–body junction of the mRNA7 species produced by mutants L1 and B1, containing either a mutant leader TRS (5′-UGAAC-3′) and a wild-type RNA body TRS or vice versa. In both cases, the TRS sequence at the mRNA7 leader–body junction was derived exclusively from the body TRS.
Figure 5
Figure 5
Model of nidovirus genome replication and sg mRNA transcription as proposed by Sawicki and Sawicki (16). Negative-strand synthesis is either continuous (a), yielding the genomic (−) strand, or discontinuous (b), yielding the sg (−) strand(s). The latter step involves translocation of the nascent (−) strand to the genomic leader TRS (c), where base pairing occurs and transcription is resumed to add the antileader sequence to the sg (−) strand. Subsequently, the sg (−) strand is used as the template for sg mRNA synthesis (d). The stem–loop structure in the leader TRS region is discussed in the text and Fig. 6.
Figure 6
Figure 6
RNA structure prediction for the EAV (+) leader TRS region, showing a striking hairpin that is conserved in arteriviruses and appears to “present” the leader TRS in its loop. Numbers refer to nucleotide positions in the EAV genome.
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