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. 2002 Aug;76(15):7632-41.
doi: 10.1128/jvi.76.15.7632-7641.2002.

Identification of an upstream sequence element required for vesicular stomatitis virus mRNA transcription

Edward E Hinzman  1 , John N Barr, Gail W Wertz
Affiliations

Identification of an upstream sequence element required for vesicular stomatitis virus mRNA transcription

Edward E Hinzman et al. J Virol. 2002 Aug.

Abstract

Vesicular stomatitis virus (VSV), the prototypic rhabdovirus, has a nonsegmented negative-sense RNA genome with five genes flanked by 3' leader and 5' trailer sequences. Transcription of VSV mRNAs is obligatorily sequential, starting from a single 3' polymerase entry site, and termination of an upstream mRNA is essential for transcription of a downstream gene. cis-acting signals for transcription of VSV mRNAs are present within the leader region, at the leader-N junction, and at the internal gene junctions. The gene junctions of VSV consist of a conserved 23-nucleotide region that includes the gene end sequence of the upstream gene, 3'-AUACU7-5', a nontranscribed intergenic dinucleotide, 3'-G/CA-5', and the gene start sequence, 3'-UUGUCNNUAG-5', at the beginning of the gene immediately downstream. Previous work has shown that the gene end sequence and intergenic region are sufficient to signal polyadenylation and termination of VSV transcripts. Mutagenesis of the gene start sequence has determined the importance of this region in the processes of initiation and 5'-end modification of mRNAs. However, because the gene end sequence is positioned directly upstream of the gene start sequence in the gene junction, and because of the requirement for termination of the upstream gene prior to transcription of the downstream gene, it has not been possible to investigate whether the gene end sequence contributes to transcription of the downstream gene. In this study, we inserted an additional gene end sequence upstream of the gene junction in a subgenomic replicon of VSV, which extended the intergenic region from 2 to 88 nucleotides. This duplication of termination signals allowed us to separate the signals required for termination from those required for initiation. We investigated the effect that the upstream gene end sequences had on downstream mRNA transcription. Our data show that the U7 tract of the upstream gene end sequence is necessary for optimal transcription of the downstream gene, independent of its role in termination of the upstream gene. Altering the sequence or changing the length of the U tract directly upstream of the gene start sequence significantly decreased transcription of the downstream gene. These results show that the U tract is a multifunctional region that is required not only for polyadenylation and termination of the upstream mRNA but also for efficient transcription of the downstream gene.

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Figures

FIG. 1.
FIG. 1.
Effect on transcription of insertion of an additional gene end signal upstream of the wild-type gene junction in a dicistronic replicon. (A) Schematic diagram of the wild-type VSV bicistronic subgenomic replicon, p8(+)NP, the dicistronic replicon having an extended IGR, pExtIG, and the RNA species transcribed. Replicon p8(+)NP (top) contained two transcriptional units separated by a single gene junction and flanked by leader and trailer regions. Replicon pExtIG (bottom) contained an additional gene end signal (shaded sequence) inserted upstream of the wild-type gene junction. Replicon pU6(GE2) (not drawn) is a mutant of pExtIG that contains a U6 tract at GE2. Abbreviations: GS, gene start sequence; GE, gene end sequence; le, leader; tr, trailer; TU, transcriptional unit; nts, nucleotides; IGR, intergenic region; R/Th, readthrough. (B) Analysis of RNAs synthesized by VSV replicons p8(+)NP (lane 1), pExtIG (lane 2), or pU6(GE2) (lane 3). Plasmids encoding the indicated replicons were transfected into cells as described in Materials and Methods. Actinomycin D-resistant RNAs were metabolically labeled with [3H]uridine, harvested, and incubated with oligo(dT) and RNase H. The resultant RNAs were analyzed by electrophoresis on agarose-urea gels, and the labeled products were visualized by fluorography. Replicon pU6(GE2) (lane 3) generated a minor product (∗), which is described in the text.
FIG. 2.
FIG. 2.
Primer extension analysis of positive-sense RNA transcribed from VSV subgenomic replicons. (A) Schematic of replicon pExtIG, positive-sense RNA products, and orientation and position of primer IGR Pext and primer R/Th Pext. (B) Primer extension analysis using end-labeled primer IGR Pext. Lanes 1 and 2, replicon p8(+)NP with (+) and without (−) VSV L, respectively; lanes 3 and 4, replicon pExtIG with (+) and without (−) VSV L, respectively; lanes 5 and 6, replicon pU6(GE2) with (+) and without (−) VSV L, respectively. Reverse transcription was carried out as described in Materials and Methods, and the cDNA products were separated by polyacrylamide gel electrophoresis. VSV L-dependent products are labeled as IG1, IG2, and IG3. A sequence ladder generated from the pExtIG template using end-labeled primer IGR Pext is shown. GE1 is indicated for reference. (C) Primer extension analysis using end-labeled primer R/Th Pext. Lane assignment is the same as for panel B. VSV L-dependent products are labeled based on comparison with data presented in panel B and a sequence ladder using pExtIG as the template and R/Th Pext as the primer (data not shown).
FIG. 3.
FIG. 3.
Analysis of RNAs transcribed by subgenomic replicons with altered U tract lengths in GE2. (A) Replicon pExtIG, mRNA products, and primer orientation are shown schematically, with the mutated region of pExtIG shaded. (B) Analysis of RNAs synthesized from replicon pExtIG (lane 5) or replicons in which the wild-type U7 tract of GE2 was increased to lengths of 14, 12, 10, or 8 U residues (lanes 1 to 4, respectively) or shortened to lengths of 6, 4, 2, or 0residues (lanes 6 to 9, respectively). RNAs were generated and analyzed as described in the legend for Fig. 1. Positions of the RNA products are indicated. (C) Primer extension analysis of RNAs synthesized from replicons described in the legend for panel A, using the end-labeled primers 1Pext and 2Pext as described in Materials and Methods. Sequence ladders were generated from the pExtIG template using the indicated end-labeled primer. Lanes are labeled according to the size of the GE2 U tract and the presence (+) or absence (−) of the VSV L support plasmid in the transfection. The positions of mRNA1, mRNA2, GE2, and GS2 are indicated. (D) Quantitation of primer extension products from three separate transfections and primer extensions, including that shown in panel C. The cDNA products were quantitated, the mRNA2/mRNA1 ratio was calculated, and this was plotted as the mean percentage of the mRNA2 synthesis from pExtIG (U7). Error bars represent one standard deviation from the mean of three primer extension experiments. Where error bars are not visible, the standard deviation was negligible.
FIG. 4.
FIG. 4.
Analysis of RNAs transcribed by subgenomic replicons with U14 tracts in either GE1 or GE2. (A) Replicon pExtIG is shown schematically with the mutated regions shaded. (B) Analysis of RNAs synthesized from replicon pExtIG (lane 6) or from replicons that contained a U14 tract at GE1 [U14(GE1); lane 1], a U14 tract at GE2[U14(GE2); lane 2], or U14 tracts at GE2 interrupted at residue 7 with either A or C (U6AU7 [lane 3] and U6CU7 [lane 4]) or at residue 8 with an A (U7AU6; lane 5). RNAs were generated and analyzed as described in the legend for Fig. 1. Positions of the RNA products are indicated. (C) Primer extension analysis of the RNAs synthesized from the replicons described in the legend for panel B. Reverse transcriptions were performed as described in Materials and Methods, using the end-labeled primers 1Pext and 2Pext. A sequence ladder generated from the pExtIG template using the end-labeled 2Pext primer is shown for reference. (D) Quantitation of primer extension products shown in panel C. The cDNA products were quantitated, and the mRNA2/mRNA1 ratio was calculated and plotted as a percentage of the mRNA2 synthesis from pExtIG.
FIG. 5.
FIG. 5.
Analysis of RNAs transcribed by subgenomic replicons with altered U tract sequences in GE2. (A) Replicon pU6(GE2) is shown schematically with the mutated region shaded. (B) Analysis of RNAs synthesized by a replicon that contained a U6 tract at GE2 [pU6(GE2); lane 1] or replicons where the U6 tract at GE2 was altered to A6 (lane 2), C6 (lane 3), G6 (lane 4), UAUAUA [(UA)3; lane 5], GCGCGC [(GC)3; lane 6], UUUAAA (U3A3; lane 7), or AAAUUU (A3U3; lane 8). RNAs were generated and analyzed as described in the legend for Fig. 1. Positions of the RNA products are indicated. (C) Primer extension analysis of the subgenomic replicons shown in panel B. Reverse transcriptions were performed as described inMaterials and Methods, using the end-labeled primers 1Pext and 2Pext. Sequence ladders were generated from the pExtIG template using the indicated end-labeled primer. (D) Quantitation of primer extension products shown in panel C. The cDNA products were quantitated, and the mRNA2/mRNA1 ratio was calculated and plotted as a percentage of the mRNA2 synthesis from pU6(GE2).
FIG. 6.
FIG. 6.
Analysis of RNAs transcribed by subgenomic replicons with mutated GE2 AUAC sequences. (A) Primer extension analysis of RNA products synthesized by subgenomic replicon pExtIG or replicons in which the 3′-AUACU7-5′ of GE2 was altered to either 3′-AUAGU7-5′, 3′-AUAAU7-5′, or the 3′-AUAC-5′ was deleted (ΔAUAC) to yield 3′-GAUGU8-5′. Reverse transcriptions were performed as described in Materials and Methods, using the end-labeled primers 1Pext and 2Pext. Sequence ladders were generated from the pExtIG template using the indicated end-labeled primer. (B) Quantitation of primer extension products shown in panel A. The cDNA products were quantitated, and the mRNA2/mRNA1 ratio was calculated and plotted as a percentage of the mRNA2 synthesis from pExtIG (3′-AUACU7-5′).
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