A vesiculovirus showing a steepened transcription gradient and dominant trans-repression of virus transcription

Abstract

Vesicular stomatitis virus (VSV) is a prototype nonsegmented, negative-sense virus used to examine viral functions of a broad family of viruses, including human pathogens. Here we demonstrate that S(2) VSV, an isolate with a small plaque phenotype compared to other Indiana strain viruses, has a transcription defect resulting in an altered pattern and rapid decline of transcription. The S(2) VSV transcription gradient is dominant over the wild-type transcription in a coinfection. This is the first characterization of an altered gradient of transcription not dependent on RNA template sequence or host response and may provide insight into new approaches to viral attenuation.

Fig 1

Growth and sequence of S₂ VSV. (A) Growth curves of wild-type and S₂ VSV at an MOI of 0.01 in BHK-21 cells. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. Dashed line indicates the minimum detection level of the assay. (B) Growth curves of wild-type and S₂ VSV at an MOI of 10 in BHK-21 cells. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. Data shown are the averages of results from three experiments with standard errors (error bars not visible at this scale). (C) Graphic representation of the sequence differences between S₂ VSV and the published VSV-San Juan sequence (GI 61833) in coding regions of the genome. Coding regions are represented as solid boxes; noncoding regions are in gray. Single-letter abbreviations are used to denote proteins encoded. Each line above the genome represents a silent mutation; each line below the genome represents a nucleotide change that results in an amino acid change.

Fig 2

RNA production in wild-type and S₂ VSV infected cells. (A) BHK cells infected with wild-type or S₂ VSV were labeled for 2 h with ³H-uridine in the presence of actinomycin D for the last 2 h of infection at times indicated. RNA from infected cells was separated on a denaturing formaldehyde gel. An autoradiograph of a representative experiment is shown. (B) Calculated molar ratios of wild-type and S₂ mRNA transcription products at 4 hpi. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. The data are averages of results from three separate experiments ± standard errors. (C) Total RNA production levels by wild-type and S₂ VSV from 4 to 10 hpi. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. The data are averages of three separate experiments ± standard errors. (D) Total RNA from BHK-21 cells mock infected (M) or infected with wild-type or S₂ VSV was separated on a denaturing formaldehyde gel, transferred to positively charged nylon, and probed using an oligonucleotide probe specific for the viral leader sequence. AG, antigenome. (E) Total RNA, as for panel D, was probed using dsDNA probes specific for each viral gene. A representative experiment is shown.

Fig 3

RNA production in singly infected or coinfected cells. (A) BHK cells infected at an MOI of 10 with wild-type, S₂, or both (total MOI of 20) viruses were labeled with ³H-uridine for 2 h at increasing times during infection and analyzed as described above. (B) Total RNA production levels in infected cells from 4 to 12 hpi. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. Coinfection indicated by open squares. (C) BHK cells were infected with wild-type VSV, S₂ VSV, or both at the MOI listed above each lane. At 8 hpi, cells were labeled with [³⁵S]methionine/cysteine for 15 min and then lysed. Total protein was separated on an SDS-PAGE gel. A representative autoradiograph is shown. The gray arrow indicates the migration of wild-type proteins; the black arrow indicates the migration of S₂ proteins.

Fig 4

RNA production by wild-type and S₂ VSV in a cell-free system. (A) In vitro transcription reactions were carried out using detergent-activated wild-type or S₂ virions, using [³²P]GTP as a trace label. RNA was purified and separated on an agarose-urea gel and exposed to a phosphorimager screen. Data shown are representative of three independent experiments. Asterisk indicates a background band seen in all reactions. (B) Calculated molar ratios of wild-type and S₂ mRNA transcription products. Wild-type VSV shown by closed circles; S₂ VSV by closed triangles. Data are averages of results from three separate experiments ± standard errors.