The immediate-early gene product encoded by open reading frame 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level - PubMed (original) (raw)

The immediate-early gene product encoded by open reading frame 57 of herpesvirus saimiri modulates gene expression at a posttranscriptional level

A Whitehouse et al. J Virol. 1998 Jan.

Abstract

The herpesvirus saimiri (HVS) immediate-early gene product encoded by open reading frame (ORF) 57 shares limited amino acid homology with HSV-1 ICP27 and Epstein-Barr virus BMLF1, both regulatory proteins. The ORF 57 gene has been proposed to be spliced based on the genome sequence, and here we confirm the intron-exon structure of the gene. We also demonstrate that a cDNA construct of the ORF 57 gene product represses the transactivating capability of the ORF 50a gene product (which is produced from a spliced transcript), but activates that of ORF 50b (an unspliced transcript). Further analyses with cotransfection experiments show that ORF 57 can either activate or repress expression from a range of both early and late HVS promoters, depending on the target gene. These results indicate that repression of gene expression mediated by the ORF 57 gene product is dependent on the presence of an intron within the target gene encoding region. Furthermore, Northern blot analysis demonstrates that the levels of mRNA transcribed from genes not containing an intron are not significantly affected in the presence of the ORF 57 gene product. This suggests that it regulates gene expression through a posttranscriptional mechanism.

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Figures

FIG. 1

FIG. 1

Response of transactivation capability of ORF 50a and ORF 50b to the ORF 57 gene product. OMK cells were transfected with 2 μg of pAWCAT2 and pAW_Hin_cII or pAW_Pst_I in the absence or presence of pRSVORF57, respectively. Cells were harvested at 48 h posttransfection, and cell extracts were assayed for CAT activity as previously described. Percentages of acetylation were calculated by scintillation counting of the appropriate regions of the chromatography plate and are shown in a graphical format, and the variations between three replicated assays are indicated.

FIG. 2

FIG. 2

Response of HVS promoters to the ORF 57 gene product. HVS promoters from the DNA polymerase gene (DNAPol), thymidine kinase gene (TK), major capsid protein gene (MCP), and glycoprotein B (gB) were cloned upstream of the CAT and β-Gal coding regions. (a) The CAT constructs contained the small t antigen intron and SV40 poly(A) region, whereas the β-Gal constructs contained only the SV40 poly(A) signal. OMK cells were transfected with pDPCAT1, pTKCAT1, pMCPCAT1, pgBCAT1, pDPLacZ1, pTKLacZ1, pMCPLacZ1, and pgBLacZ1 in the absence or presence of pRSVORF57. (b) The CAT constructs contained only the SV40 poly(A) region, whereas the β-Gal constructs contained the small t antigen intron and the SV40 poly(A) signal. OMK cells were transfected with pDPCAT2, pTKCAT2, pMCPCAT2, pgBCAT2, pDPLacZ2, pTKLacZ2, pMCPLacZ2, and pgBLacZ2 in the absence or presence of pRSVORF57. Cells were harvested at 48 h posttransfection, and cell extracts were assayed for CAT and β-Gal activity. Percentages of CAT acetylation were calculated by scintillation counting of the appropriate regions of the chromatography plate. The activity of β-Gal was determined by the rate of production of _ortho_-nitrophenol from the substrate _ortho_-nitrophenyl galactoside. Results are shown in graphical format, and the variations between three replicated assays are indicated.

FIG. 3

FIG. 3

Northern blot analysis of HVS promoter CAT and β-Gal constructs in response to the ORF 57 gene product. RNA was isolated from OMK cells transfected with the constructs pRSVORF57 (lane 1), pTKCAT1 (lane 2), pTKCAT2 (lane 3), pTKCAT1 and pRSVORF57 (lane 4), or pTKCAT2 and pRSVORF57 (lane 5) (a) plus pgBCAT (b), pTKLacZ (c), and pgBLacZ (d), separated by electrophoresis on a 1% denaturing formaldehyde agarose gel, blotted onto a nylon membrane, and hybridized with radiolabelled probes specific for the CAT and β-Gal coding regions.

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