Overlapping signals for transcription and replication at the 3' terminus of the vesicular stomatitis virus genome

Abstract

Transcription and replication signals within the negative-sense genomic RNA of vesicular stomatitis virus (VSV) are located at the 3' terminus. To identify these signals, we have used a transcription- and replication-competent minigenome of VSV to generate a series of deletions spanning the first 47 nucleotides at the 3' terminus of the VSV genome corresponding to the leader gene. Analysis of these mutants for their ability to replicate showed that deletion of sequences within the first 24 nucleotides abrogated or greatly reduced the level of replication. Deletion of downstream sequences from nucleotides 25 to 47 reduced the level of replication only to 55 to 70% of that of the parental template. When transcription activity of these templates was measured, the first 24 nucleotides were also found to be required for transcription, since deletion of these sequences blocked or significantly reduced transcription. Downstream sequences from nucleotides 25 to 47 were necessary for optimal levels of transcription. Furthermore, replacement of sequences within the 25 to 47 nucleotides with random heterologous nonviral sequences generated mutant templates that replicated well (65 to 70% of the wild-type levels) but were transcribed poorly (10 to 15% of the wild-type levels). These results suggest that the minimal promoter for transcription and replication could be as small as the first 19 nucleotides and is contained within the 3'-terminal 24 nucleotides of the VSV genome. The sequences from nucleotides 25 to 47 may play a more important role in optimal transcription than in replication. Our results also show that deletion of sequences within the leader gene does not influence the site of transcription reinitiation of the downstream gene.

FIG. 1

(A) The plasmid p9BN encoding the antigenomic plus-sense minireplicon. Only the relevant regions of the plasmid are shown. φ10, Tφ, and δ represent the T7 RNA polymerase promoter, the terminator, and the hepatitis delta virus ribozyme sequences, respectively; le′ and tr′ represent complementary sequences of leader and trailer regions of the VSV genome; N and ΔL represent the coding sequences of the N and part of the L gene of VSV. In plasmid-transfected cells, the antigenomic minireplicon, synthesized from p9BN by T7 RNA polymerase, is encapsidated by the viral N protein to generate plus-sense nucleocapsid with two additional guanosine residues at the 5′ terminus. Replication of this nucleocapsid by VSV RNA polymerase generates the genomic minus-sense nucleocapsid, which serves as the template for transcription by VSV RNA polymerase to synthesize NΔL mRNA and also for replication to generate the antigenomic plus-sense nucleocapsid. The plus-sense RNA synthesized by VSV RNA polymerase differs from the plus-sense RNA synthesized by T7 RNA polymerase by the absence of the two 5′-terminal guanosine residues. (B) Various mutant minireplicons with deletion or substitution of nucleotides (as shown) at the 5′ terminus of the antigenomic minireplicon (9BN), which corresponds to the leader gene sequences at the 3′ terminus of genomic-sense RNA. Sequences of the first 55 nucleotides at the 5′ terminus of the antigenomic RNA are shown. They correspond to 47 nucleotides of leader RNA sequence, three nontranscribed intergenic nucleotides (UUU, bold-faced), and the first five nucleotides (AACAG, outlined) of N mRNA. Underlined sequences represent nucleotides that are complementary to the sequences at the 3′ terminus of the antigenome. Deleted nucleotides within the leader region are represented by dots. Random heterologous sequences that replace leader sequences are shown in boxes.

FIG. 2

(A) Replication of various mutant antigenomic minireplicons to generate genomic-sense 9BN(−) RNA. Cells infected with vTF7-3 were transfected with plasmids encoding the N, P, and L (or without L in the negative control, lane 2) proteins and either p9BN or deletion mutant plasmids. At 16 h posttransfection, cells were labeled for 6 h with [³H]uridine in the presence of actinomycin D. Replicated RNAs in the nucleocapsids were immunoprecipitated, purified, and analyzed by agarose-urea gel electrophoresis as described in Materials and Methods. G and N represent the G mRNA and N mRNA of VSV isolated from infected cells. The migration position of plus-sense RNA is indicated by an arrow. 9BN(−) is the negative-sense replication product. An asterisk indicates the band of NΔL mRNA, which is sometimes immunoprecipitated (30, 50) by anti-VSV antibodies. (B) Relative levels of replication of deletion mutant minireplicons to produce genomic-sense products. Histograms show averages and range of levels of replication of various deletion mutants (described at the top of panel A) from three independent experiments.

FIG. 3

(A) Primer extension analysis to detect 5′ termini of various plus-sense RNA products in transfected cells. Lane 6, extension products of RNAs from cells after transcription and replication; lane 5, extension products of RNAs without transcription or replication. The extension products labeled a, b, and c are described in the text. The 5′ terminus of the replication product (indicated by a dot in lane 6) maps to the T residue (identified by the top arrow on the left) corresponding to the first nucleotide (U) in the VSV genome. The 5′ terminus of the NΔL mRNA transcription product maps to the first T residue (identified by the bottom arrow on the left) of the transcription initiation signal UUGUC in the VSV genome. The more intense top bands in doublets c and b most likely represent the extension product of capped mRNA. (B) Analysis of plus-sense RNA replication products from mutant templates by primer extension. Replication products are identified by dots in the lanes. Only the top portion of the gel is shown. (C) Average normalized replication of various mutants (shown at the top of panel B) from two separate experiments as determined with the following formula: Normalized levels of plus-sense replication products (%) = relative levels of plus-sense replication products/relative levels of minus-sense template × 100.

FIG. 4

(A) Analysis of transcription from the mutant templates. Cells were infected with vTF7-3 and transfected with plasmids encoding N, P, and L and wild-type or mutant minireplicons. At 16 h posttransfection, cells were treated with actinomycin D and labeled with [³H]uridine for 6 h. Total labeled RNA from these cells was analyzed by electrophoresis in agarose-urea gel. Lane 2 shows RNA from the negative control (no L plasmid in transfection) sample. An arrow indicates the NΔL mRNA transcription product. (B) Normalized levels of transcription from the mutant templates. Values were obtained by using the formula: Nomarlized transcription (%) = relative levels of transcription/relative levels of minus-sense template × 100. Histograms represent the averages and ranges of values from three separate experiments.

FIG. 5

Transcription and replication activities of larger deletion mutant templates. (A) Analysis of negative-sense replication products [p9BN(−)] as described in Fig. 2A. Lane 2 shows replication products from the negative control (no L plasmid in transfection) sample. (B) Analysis of plus-sense replication products (identified by dots in lanes 2 to 4) by primer extension analysis as described in the legend for Fig. 3B. (C) Analysis of NΔL mRNA transcription products (indicated by arrow) as described in the legend for Fig. 4A. (D) Normalized levels of transcription and replication from minus-sense templates, as described in the legends for Fig. 3C and 4B.

FIG. 6

Transcription and replication activities of substitution mutant minireplicons. (A) Analysis of minus-sense replication products (indicated by arrow) as described in the legend for Fig. 2A. Lane 1 contains RNA from the negative control (no L) sample. (B) Analysis of NΔL mRNA transcription product (indicated by arrow) as described in the legend for Fig. 4A. (C) Normalized levels of transcription and replication from negative-sense templates as described in the legends for Fig. 3C and 4B.

FIG. 7

Primer extension analysis to examine the 5′ terminus of transcription products from the mutant minireplicons. Analysis was performed as described in the legend for Fig. 3A, and the data shown in the figure represent the extension products of NΔL mRNA. Lane 5 shows the extension products of RNA from a negative control experiment in which L plasmid was omitted from the transfection mixture. The 5′ terminus of uncapped NΔL mRNA from wild-type and mutant minireplicon templates maps to the T residue (shown by the arrow in the sequence ladder) of the transcription initiation site, UUGUC, in the viral genome.