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. 2004 Aug;78(15):8102-13.
doi: 10.1128/JVI.78.15.8102-8113.2004.

Regulation of relative abundance of arterivirus subgenomic mRNAs

Alexander O Pasternak  1 , Willy J M Spaan, Eric J Snijder
Affiliations

Regulation of relative abundance of arterivirus subgenomic mRNAs

Alexander O Pasternak et al. J Virol. 2004 Aug.

Abstract

The subgenomic (sg) mRNAs of arteriviruses (order Nidovirales) form a 5'- and 3'-coterminal nested set with the viral genome. Their 5' common leader sequence is derived from the genomic 5'-proximal region. Fusion of sg RNA leader and "body" segments involves a discontinuous transcription step. Presumably during minus-strand synthesis, the nascent RNA strand is transferred from one site in the genomic template to another, a process guided by conserved transcription-regulating sequences (TRSs) at these template sites. Subgenomic RNA species are produced in different but constant molar ratios, with the smallest RNAs usually being most abundant. Factors thought to influence sg RNA synthesis are size differences between sg RNA species, differences in sequence context between body TRSs, and the mutual influence (or competition) between strand transfer reactions occurring at different body TRSs. Using an Equine arteritis virus infectious cDNA clone, we investigated how body TRS activity affected sg RNA synthesis from neighboring body TRSs. Flanking sequences were standardized by head-to-tail insertion of several copies of an RNA7 body TRS cassette. A perfect gradient of sg RNA abundance, progressively favoring smaller RNA species, was observed. Disruption of body TRS function by mutagenesis did not have a significant effect on the activity of other TRSs. However, deletion of body TRS-containing regions enhanced synthesis of sg RNAs from upstream TRSs but not of those produced from downstream TRSs. The results of this study provide considerable support for the proposed discontinuous extension of minus-strand RNA synthesis as a crucial step in sg RNA synthesis.

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Figures

FIG. 1.
FIG. 1.
(A) Schematic diagram of the genome organization and expression of EAV, the arterivirus prototype. The regions of the genome specifying the leader (L) sequence, the replicase gene (ORFs 1a and 1b), and the structural protein genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2 to 7) is depicted below. The black boxes in the genomic RNA indicate the positions of leader and major body TRSs. (B) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus-strand or minus-strand RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus-strand template. For details, see the text. (C) Northern hybridization analysis of intracellular EAV RNA resolved by denaturing agarose gel electrophoresis. As a probe, 32P-labeled oligonucleotide E154 was used, which is complementary to the 3′ end of all viral plus-strand RNA molecules (see Materials and Methods).
FIG. 2.
FIG. 2.
(A) Scheme of constructs with repeated RNA7 body TRS cassettes. The upper panel shows a close-up view of the 3′-proximal quarter of the EAV genome, where the structural gene ORFs (2a to 7) and body TRSs are located. The TRSs are indicated with triangles. The nested set of sg mRNAs, including the two major subspecies of sg mRNA3 (33), is shown below. The contents of the TRS7 cassette (see Materials and Methods) are depicted above. The lower panel shows the composition of the constructs with one (mA) to four (mABCD) repeats of the TRS7 cassette. For each construct, the corresponding sg RNAs are shown. In all constructs, the RNA2 TRS was knocked out by mutation (depicted by crossed squares). (B) Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type (wt) EAV infectious cDNA clone or from the constructs with repeated TRS7 cassettes. (C) Amounts of genomic and sg mRNAs produced by the wild-type EAV construct and by the TRS7 repeat constructs. The average (of two experiments) amounts of RNAs are shown.
FIG. 3.
FIG. 3.
(A) Scheme of seven mutant three-repeat constructs, representing all possible combinations of a wild-type (wt) body TRS and a mutant body TRS. The wild-type TRSs are indicated by triangles and capital letters, and the mutant TRSs are indicated by crossed squares and lowercase letters. (B) Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from the wild-type and seven mutant three-repeat constructs of the first series (TRS mutations 5′-UCAACU-3′ to 5′-UgAAgU-3′). (C) Relative amounts of genomic and sg mRNAs produced by the mutant three-repeat constructs. The amounts of each RNA species (genomic and subgenomic) produced by the mutant constructs were independently related to the amounts of the corresponding RNA species produced by the wild-type three-repeat construct (mABC), which were set at 100%. The average (of three experiments) amounts of RNAs are shown.
FIG. 4.
FIG. 4.
(A) Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type (wt) EAV infectious cDNA clone or from the wild-type and seven mutant three-repeat constructs of the second series (TRS mutations 5′-UCAACU-3′ to 5′-aguACa-3′). (B) Relative amounts of genomic and sg mRNAs produced by the mutant three-repeat constructs of the second series. See the legend to Fig. 3 for the calculation of RNA levels. The average (of three experiments) amounts of RNAs are shown.
FIG. 5.
FIG. 5.
Schematic representation of the proposed homologous recombination in the TRS7 repeat constructs. For clarity, mABc is shown as a parental construct. Both upper panels show its composition as well as the sg mRNA molecules (A and B) produced. Synthesis of the smallest sg mRNA molecule (RNA C) is knocked out by TRS mutation in the parental construct. Homologous recombination may result in deletion of either one (A) or two (B) TRS cassettes, rendering the sg mRNAs one (Δc recombinant) or two (ΔBc recombinant) cassettes smaller, respectively, as depicted in the lower panels. The sizes of sg mRNAs B (Δc recombinant) and A (ΔBc recombinant) would match the size of sg mRNA C, which would appear to be partially restored. Analogous homologous recombination may result in insertion of one or two cassettes.
FIG. 6.
FIG. 6.
(A) Scheme of the deletion constructs (30). See the text for details. (B) Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from the deletion constructs. The RNA2 bands are boxed. (C) Relative amounts of genomic and sg mRNAs produced by the deletion constructs. Amounts of each RNA species (genomic and subgenomic) produced by the deletion constructs were independently related to the amounts of the corresponding RNA species produced by the wild-type construct, which were set at 100%. The average (of three experiments) amounts of RNAs are shown.
FIG. 7.
FIG. 7.
(A) Scheme of the constructs bearing TRS-free antisense EAV sequences. The antisense sequences were inserted into either the NcoI restriction site (030-1817f) or between the BssHII and MluI restriction sites (030-1762f). Note the reverse orientations of the body TRSs 3.1, 3.2, 4, and 5 in these constructs. The corresponding deletion constructs (030-1817 and 030-1762) are also shown. (B) Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from the TRS-free insertion and corresponding deletion constructs. The RNA2 bands are boxed. (C) Relative amounts of genomic and sg mRNAs produced by the TRS-free insertion and corresponding deletion constructs. See the legend to Fig. 6 for the calculation of RNA levels. The average (of two experiments) amounts of RNAs are shown.
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