Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III - PubMed (original) (raw)

Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III

M Ahmed et al. J Virol. 1998 Oct.

Abstract

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)- RNA synthesis were measured by incorporation of [3H]uridine. v6 and tsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)- RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and alpha-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.

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Figures

FIG. 1

FIG. 1

Poly(A)+ (A) and poly(A)− (B) RNA synthesis in BHK cells infected with wt, _ts_O82, and v6 viruses. BHK cells were infected with wt (open circles), _ts_O82 (closed squares), and v6 (closed triangles) viruses at a multiplicity of infection of 20 PFU/cell. Parallel samples were infected in the presence of actinomycin D. At 2, 4, and 6 h postinfection, cells were labeled with [3H]uridine (20 μCi/ml) for 30 min. Cells were harvested and lysed in SDS lysis buffer. To separate poly(A)+ and poly(A)− RNAs, lysates were incubated in the presence of oligo(dT) cellulose (Invitrogen), washed in high-salt buffer, and eluted in low-salt buffer. Samples were then precipitated with 7% trichloroacetic acid on ice and washed twice with 7% trichloroacetic acid. Acid-precipitable radioactivity was measured by scintillation counting. Values of samples incubated in the presence of actinomycin D were subtracted from the total counts to determine the rate of host RNA synthesis. Data shown are means ± standard deviations for four experiments.

FIG. 2

FIG. 2

Effect of M protein on transcriptional activity of genes dependent on RNAPI. (A) BHK cells were cotransfected with pHrMr plasmid DNA and 0, 36, or 360 ng of in vitro-transcribed wt M mRNA. Cells that received no M mRNA were cotransfected with 360 ng of yeast RNA as a negative control. At 24 h posttransfection, nuclei were isolated and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to linearized pHrMr plasmid DNA fixed on nitrocellulose membrane filters. (B) BHK cells were cotransfected with pHrMr plasmid DNA and 0 or 360 ng of _ts_O82 M mRNA. Transcription of pHrMr DNA was assayed by nuclear runoff analysis as described above for panel A. (C) BHK cells were transfected with pHrMr DNA or no plasmid DNA as a control for the specificity of hybridization. Transcription of pHrMr DNA was assayed by nuclear runoff analysis as described above for panel A. (D) BHK cells were infected at a multiplicity of infection of 20 PFU/cell with wt or _ts_O82 virus. Mock-infected cells were used as a control. Nuclei were isolated 6 h postinfection, and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to a cDNA fragment of 18S rRNA immobilized on nitrocellulose membranes. (E) The data from four (A and D) or three (B) separate experiments were quantitated by densitometry and expressed as a percentage of the control without M mRNA for the transfection experiments and as a percentage of the uninfected control for the virus-infected cells. The data are means ± standard deviations.

FIG. 3

FIG. 3

Effect of M protein on transcriptional activity of genes dependent on RNAPII. (A) BHK cells were cotransfected with pSV2.CAT plasmid DNA and 0, 36, or 360 ng of in vitro-transcribed wt M mRNA. Cells that received no M mRNA were cotransfected with 360 ng of yeast RNA as a negative control. At 24 h posttransfection, nuclei were isolated and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to linearized pSV2.CAT plasmid DNA fixed on nitrocellulose membrane filters. (B) BHK cells were cotransfected with pSV2.CAT plasmid DNA and 0 or 360 ng of _ts_O82 M mRNA. Transcription of pSV2.CAT DNA was assayed by nuclear runoff analysis as described above for panel A. (C) BHK cells were transfected with pSV2.CAT DNA or no plasmid DNA as a control for the specificity of hybridization. Transcription of pSV2.CAT DNA was assayed by nuclear runoff analysis as described above for panel A. (D) BHK cells were infected at a multiplicity of infection of 20 PFU/cell with wt or _ts_O82 virus. Mock-infected cells were used as a control. Nuclei were isolated 6 h postinfection, and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to a cDNA fragment of α-tubulin mRNA immobilized on nitrocellulose membranes. (E) The data from four (A and B) or two (D) separate experiments were quantitated by densitometry and expressed as a percentage of the control without M mRNA for the transfection experiments and as a percentage of the uninfected control in the case of the virus-infected cells. The data are means ± standard deviations.

FIG. 4

FIG. 4

Effect of M protein on transcriptional activity of adenovirus VA genes dependent on RNAPIII. (A) BHK cells were cotransfected with pAdVantage (pAdV) plasmid DNA and 0, 36 or 360 ng of in vitro-transcribed wt M mRNA. Cells that received no M mRNA were cotransfected with 360 ng of yeast RNA as a negative control. At 24 h posttransfection, nuclei were isolated and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to linearized pAdV plasmid DNA fixed on nitrocellulose membrane filters. (B) BHK cells were cotransfected with pAdV plasmid DNA and 0 or 360 ng of _ts_O82 M mRNA. Transcription of pAdV DNA was assayed by nuclear runoff analysis as described above for panel A. (C) BHK cells were transfected with pAdV DNA or no plasmid DNA as a control for the specificity of hybridization. Transcription of pAdVantage DNA was assayed by nuclear runoff analysis as described above for panel A. (D) The data from four (A) or three (B) separate experiments were quantitated by densitometry and expressed as a percentage of the control without M mRNA. The data are means ± standard deviations.

FIG. 5

FIG. 5

Effect of M protein on transcriptional activity of 5S rRNA genes dependent on RNAPIII. (A) BHK cells were transfected with 0 or 360 ng of in vitro-transcribed wt M mRNA. At 24 h posttransfection, nuclei were isolated and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to linearized cDNA of 5S rRNA fixed on nitrocellulose membrane filters. (B) BHK cells were infected at a multiplicity of infection of 20 PFU/cell with wt or _ts_O82 virus. Mock-infected cells were used as a control. Nuclei were isolated 6 h postinfection, and RNA transcripts were elongated in the presence of [α-32P]UTP. Labeled RNAs were isolated and hybridized to a cDNA of 5S rRNA immobilized on nitrocellulose membranes. (C) The data from four separate experiments were quantitated by densitometry and expressed as a percentage of the control without M mRNA for the transfection experiments and as a percentage of the uninfected control in the case of the virus-infected cells. The data are means ± standard deviations and are plotted on a logarithmic scale to accommodate all of the values.

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References

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