The VSV polymerase can initiate at mRNA start sites located either up or downstream of a transcription termination signal but size of the intervening intergenic region affects efficiency of initiation

Abstract

Transcription by the vesicular stomatitis virus (VSV) polymerase has been characterized as obligatorily sequential with transcription of each downstream gene dependent on termination of the gene immediately upstream. In studies described here we investigated the ability of the VSV RNA-dependent RNA polymerase (RdRp) to access mRNA initiation sites located at increasing distances either downstream or upstream of a transcription termination signal. Bi-cistronic subgenomic replicons were constructed containing progressively extended intergenic regions preceding the initiation site of a downstream gene. The ability of the RdRp to access the downstream sites was progressively reduced as the length of the intergenic region increased. Alternatively, bi-cistronic replicons were constructed containing an mRNA start signal located at increasing distances upstream of a termination site. Analysis of transcription of these "overlapped" genes showed that for an upstream mRNA start site to be recognized it had to contain not only the canonical 3'-UUGUCnnUAG-5' gene start signal, but that signal needed also to be preceded by a U7 tract. Access of these upstream mRNA initiation sites by the VSV RdRp was proportionately reduced with increasing distance between the termination site and the overlapped initiation signal. Possible mechanisms for how the RdRp accesses these upstream start sites are discussed.

Figure 1. Analysis of the transcription activity of bicistronic genome analogs with lengthened intergenic regions (IGRs)

(A). Schematic representation of plasmid pExt-IG and its derivatives. These plasmids were individually transfected into vTF7-3 infected BHK cells along with support plasmids expressing VSV N, P and L ORFs, where they expressed an RNA representing a VSV anti-genomic analog. Encapsidation and replication of this RNA yielded a genome analog containing upstream (mRNA1) and downstream (mRNA2) transcriptional units separated by IGRs ranging between 2 and 268 nucleotides in length. Locations of U7 tract and intergenic di-nucleotide (di-nt) 3’ of the mRNA 2 initiation signal are shown. (B). Abundance of mRNA1 and mRNA2 transcribed from bicistronic templates was determined by dual extension of end-labeled oligonucleotides 1Pext and 2Pext, respectively. RNAs generated from template pExt-IG88 were also subjected to single primer extension reactions using individually 1Pext and 2Pext, to show position of mRNA 1 and mRNA2 respectively. Extension products were electrophoresed on a denaturing 6% acrylamide sequencing gel, and visualized by autoradiography. (C). The ability of the VSV RdRp to access the downstream gene start was calculated by quantitation of radiolabeled products representing downstream mRNA2 as a fraction of upstream mRNA1. This figure was then expressed as a percentage of that determined for template IGR2, which has the wild-type IGR length of 2 nts, and plotted as closed circles. Templates having the sub-optimal IGR start sites removed by mutagenesis were analyzed in the same way, and the values for mRNA2/mRNA1 % of IGR2 are also plotted as open diamonds.

Figure 2. Analysis of the transcription activity of bicistronic genome analogs having genes that overlap by 24 nucleotides

(A). Schematic representation of genome analog p8(+)NP and the mRNAs that are transcribed from upstream (mRNA1) and downstream (mRNA2) genes separated by the wild-type gene junction. (B). Schematic representation of genome analogs M24-GS and M24-U7, in which the mRNA2 initiation signal is located 24 nts upstream of the mRNA1 termination signal. M24-U7 differs from M24-GS in that it contains an U7 tract upstream of the transcriptional initiation site. Additional template M24-A7 was also generated in which the U7 tract was substituted for A7. (C). These bicistronic genome analogs were expressed in BHK cells, and their RNA synthesis activity compared to that of wild-type template p8(+)NP, which has conventional non-overlapping genes. Actinomyin D-resistant RNAs were metabolically labeled with 3H uridine, harvested, treated with RNase H/oligo d(T) and then electrophoresed on a denaturing 1.75% agarose-urea gel. RNAs were visualized by autoradiography and the positions of mRNAs 1 and 2 are marked with arrowheads. (D). The RNA synthesis activity of bicistronic templates was also analyzed by primer extension analysis using end-labeled oligonucleotide 1434, which anneals within the 5’ end of mRNA 2, shown schematically in panels A and B. Alignment of the 1434 extension product with the adjacent sequence ladder confirms the mRNA2 product of template M24-U7 is initiated at the overlapped initiation site.

Figure 3. Visualization of RNAs transcribed from bicistronic genome analogs with overlapping genes

(A). Schematic representation of genome analogs in which the mRNA 2 initiation signal was positioned at increasing distances upstream of the mRNA1 termination signal, up to a distance of 86 nucleotides. Transcription products mRNA1 and mRNA2 are also shown schematically. (B). Bicistronic genome analogs were generated in BHK cells, and their RNA synthesis activity compared to that of wild-type template p8(+)NP, which has conventional nonoverlapping genes. Actinomycin D-resistant RNAs were metabolically labeled with 3H uridine, harvested, treated with RNase H/oligo d(T) and then electrophoresed on a denaturing 1.75% agarose-urea gel. RNAs were visualized by autoradiography and the positions of mRNAs 1 and 2 are marked with arrowheads.

Figure 4. Analysis of RNAs transcribed from bicistronic genome analogs with overlapping genes using primer extension

(A). Schematic representation of genome analogs in which the mRNA 2 initiation signal was positioned at increasing distances upstream of the mRNA1 termination signal, up to a distance of 97 nucleotides. Transcription products are shown schematically, including the 3’ truncated form of mRNA 2 which is transcribed from templates having overlaps over 56 nts, due to activity of the mRNA1 termination signal. (B). RNAs generated by these bicistronic genome analogs were harvested from BHK cells and analyzed by primer extension analysis using end-labeled oligonucleotide 1434, which anneals within the middle region of mRNA2, shown schematically in panel A. Abundance of each mRNA 2 species is shown as a percentage of mRNA 2 levels generated by template p8+NP and represents the mean of at least 3 separate experiments. The cDNA expressing wild-type anti-genome analog 8(+)NP was sequenced with primer 1434 to act as size marker. (C). RNAs generated from genome analogs having overlaps in excess of 56 nts were also analyzed by primer extension using end-labeled oligonucleotide 1365, which annealed at the 5’ end of mRNA2 and allowed detection of mRNA2 transcripts that terminated at the mRNA1 termination signal, shown schematically in panel A. As above, abundance of each mRNA 2 species is shown as a percentage of mRNA 2 levels generated by template M66, and represents the mean of at least 3 separate experiments.

Figure 5. Investigating the upper limit of RdRp access to an overlapped initiation signal using primer extension analysis

(A). Schematic representation of genome analogs that possess an mRNA 2 initiation signal positioned at increasing distances upstream of the mRNA1 termination signal, up to a distance of 200 nucleotides. The mRNA1 and mRNA2 transcription products are also shown schematically, all of which are predicted to terminate at the mRNA1 termination signal. (B). RNAs generated by templates M97, M122, M150 and M200 were harvested from BHK cells and analyzed by primer extension analysis using end-labeled oligonucleotide T7–14, which anneals within the 3’ end of mRNAs 1 and 2, shown schematically in panel A. Bands representing mRNA2-templated extension products from five separate experiments were quantified and their abundance shown as a percentage of mRNA1 levels from the same template. Percent abundance relative to template M97 was also determined.