Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Dec 17;20(24):7220-8.
doi: 10.1093/emboj/20.24.7220.

Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis

A O Pasternak  1 , E van den Born, W J Spaan, E J Snijder
Affiliations

Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis

A O Pasternak et al. EMBO J. .

Abstract

Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5' end of the genome fused to different sequences ('bodies') derived from the 3' end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader-body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis.

PubMed Disclaimer

Figures

None
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 genes are indicated. The nested set of EAV mRNAs (genome and sg mRNAs 2–7) is depicted below. The black boxes in the genomic RNA indicate the position of leader and major body TRSs. (B and C) Alternative models for nidovirus discontinuous sg RNA synthesis. The discontinuous step may occur during either plus strand (B) or minus strand (C) RNA synthesis. In the latter case, sg mRNAs would be synthesized from an sg minus strand template. For details see text.
None
Fig. 2. Northern analysis of EAV-specific RNA isolated from cells transfected with RNA transcribed either from the wild-type EAV infectious cDNA clone or from TRS pentanucleotide mutants (UCAAC to AGUUG). The results of two independent experiments are shown.
None
Fig. 3. Relative amounts of sg RNA7 produced by EAV TRS mutants. For each set of transfections (representing one experiment), the EAV wild-type construct pEAV030 (van Dinten et al., 1997) was taken along as a positive control. For every mutant, the level of sg RNA7 synthesis was calculated as [(sg/g)/(sg/g)wt] × 100%: it was corrected for the level of genomic RNA (used as an internal standard; see text) and subsequently was related to the level of sg RNA7 produced by the wild-type control in the same experiment, which was also corrected for the corresponding genomic RNA level. The relative sg RNA7 level of the wild-type control was set at 100%.
None
Fig. 4. (A) Northern analysis of EAV-specific RNA isolated from cells transfected with wild-type EAV RNA or 11 selected TRS mutants. (B) Relative amount of sg RNA7 produced by the selection of 11 TRS mutants. See the legend to Figure 3 for the calculation of sg RNA7 levels. For each construct in (B), experiments were repeated at least three times and average sg RNA levels are shown.
None
Fig. 5. Sequence analysis of mRNA7 leader–body junctions from position 1 TRS mutants. Sequences were determined directly from sg mRNA7-specific RT–PCR products. For the U1A and U1C mutants, the sequence shown corresponds to the plus strand of sg RNA7. For sequencing-related technical reasons, the minus strand sequence was determined for the U1G mutants; a mirror image of the electrophero gram is shown with the corresponding plus strand sequence listed at the top of the panel. For every mutant, a sequence alignment of the leader (red) and body (blue) TRSs and surrounding sequences is shown (TRSs are boxed). The mRNA7 leader–body junctions detected by our sequence analysis are shown in yellow.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aaziz R. and Tepfer,M. (1999) Recombination in RNA viruses and in virus-resistant transgenic plants. J. Gen. Virol., 80, 1339–1346. - PubMed
    1. Adkins S., Siegel,R.W., Sun,J.H. and Kao,C.C. (1997) Minimal templates directing accurate initiation of subgenomic RNA synthesis in vitro by the brome mosaic virus RNA-dependent RNA polymerase. RNA, 3, 634–647. - PMC - PubMed
    1. Almazan F., Gonzalez,J.M., Penzes,Z., Izeta,A., Calvo,E., Plana-Duran.J. and Enjuanes,L. (2000) Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc. Natl Acad. Sci. USA, 97, 5516–5521. - PMC - PubMed
    1. Baker S.C. and Lai,M.M.C. (1990) An in vitro system for the leader-primed transcription of coronavirus mRNAs. EMBO J., 9, 4173–4179. - PMC - PubMed
    1. Baric R.S. and Yount,B. (2000) Subgenomic negative-strand RNA function during mouse hepatitis virus infection. J. Virol., 74, 4039–4046. - PMC - PubMed

Publication types