Selection for gene junction sequences important for VSV transcription

Abstract

The heptauridine tract at each gene end and intergenic region (IGR) at the gene junctions of vesicular stomatitis virus (VSV) have effects on synthesis of the downstream mRNA, independent of their respective roles in termination of the upstream mRNA. To investigate the role of the U tract and the IGR in downstream gene transcription, we altered the N/P gene junction of infectious VSV such that transcription levels would be affected and result in altered molar ratios of the N and P proteins, which are critical for optimal viral RNA replication. The changes included extended IGRs between the N and P genes and shortening the length of the heptauridine tract upstream of the P gene start. Viruses having various combinations of these changes were recovered from cDNA and selective pressure for efficient viral replication was applied by sequential passage in cell culture. The replicative ability and sequence at the altered intergenic junctions were monitored throughout the passages to compare the effects of the changes at the IGR and U tract. VSV variants with wild-type U tracts upstream of the P gene replicated to levels similar to wt VSV. Variants with shortened U tracts were reduced in their ability to replicate. With passage, populations emerged that replicated to higher levels. Sequence analysis revealed that mutations had been selected for in these populations that increased the length of the U tract. This correlated with an increase in abundance of P mRNA and protein to provide improved N:P protein molar ratios. Extended IGRs resulted in decreased downstream transcription but the effect was not as extensive as that caused by shortened U tracts. Extended IGRs were not selected against in 5 passages. Our results indicate that the size of the upstream gene end U tract is an important determinant of efficient downstream gene transcription in infectious virus.

FIG. 1

Schematic diagram of wt VSV and variant viruses engineered to contain extended IGRs and U tract alterations at the N/P gene junction. The indicated sequences were inserted between the N gene end sequence and the P gene start sequence of the cDNA clone of VSV, and virus was rescued as described in Materials and Methods. The inserted gene end sequence is shown in bold, and the Xho I restriction site underlined. Abbreviations: le, leader; tr, trailer; WT, wild type; nts, nucleotides.

FIG. 2

Mean plaque diameters for wt VSV and variant viruses following sequential passage. Virus from passage 1 (A), 3 (B), and 5 (C) were analyzed. Viral plaque diameters were measured directly from plaques formed on Vero-76 cell monolayers. The mean plaque diameter for each virus was determined, normalized to wt VSV at each passage, and expressed as the mean plaque diameter with a 95% confidence interval.

FIG. 3

Single step growth analysis of wt VSV and variant viruses. BHK-21 cells infected with indicated viruses at an MOI of 5 at 37C. Virus from passage 1 (A), 3 (B), and 5 (C) were analyzed. Samples of the supernatant medium were harvested at the indicated time points and titrated in duplicate by plaque assay on Vero-76 cells.

FIG. 4

Sequence analysis of the N/P gene junction of the indicated viruses at passage 1, 3, and 5. (A) Diagram of the VSV genome and relative positions of primers used in RT – PCR analysis. (B and C) Sequence chromatogram traces of bulk RT – PCR products from U6-21 (B) and U6-106 (C) genomes at the indicated passage. The N gene end sequence (N GE), intergenic region (IGR), and P gene start sequence (P GS) are indicated above the chromatograms. Underlined sequence indicates potential change(s) from the passage 1 sequence.

FIG. 5

Comparison of the P:N mRNA ratios for wt VSV and variant viruses at passages 1, 3, and 5. (A) Schematic diagram of the VSV genome, the N and P mRNAs, and the relative positions of primers N-Pext and P-Pext that anneal to the N and P mRNA, respectively, and also to corresponding positions within the anti-genome. (B) Primer extension analysis of the N and P mRNAs from the indicated viruses at the indicated passage using end-labeled primers N-Pext and P-Pext as described in Materials and Methods. The position of the products corresponding to the antigenome, the N mRNA, and the P mRNA are indicated at the left of the figure. (C) Quantitation of primer extension products from three independent experiments, including that shown in panel B. The P:N mRNA ratio was determined, normalized to the wt VSV P: N mRNA ratio at that passage, and expressed as the mean percentage relative to wt VSV. Error bars represent the standard error of the mean. Where error bars are not visible, the standard error was negligible.

FIG. 6

Comparison of the N/P protein molar ratios for wt VSV and variant viruses at passage 1, 3, and 5. (A) Viral proteins synthesized in BHK-21 cells infected with the indicated viruses at the specified passage. Protein was labeled with [35S]methionine as described in Materials and Methods, resolved by low bis PAGE, and detected by autoradiography. The virus and passage number are indicated at the top and the viral proteins are identified at the left of the figure. Mock 1 and 2 are uninfected cells labeled as described above. (B) P – N protein molar ratios from three independent experiments, including that shown in panel A. Molar ratios were calculated after normalizing for methionine content of the P and N proteins and expressed as a mean percentage of the wt VSV P:N ratio for the given passage. Error bars represent the standard error of the mean. (C) M:N protein molar ratios, determined and plotted as described in the legend for panel B.