The open reading frame (ORF) 50a gene product regulates ORF 57 gene expression in herpesvirus saimiri

Abstract

We have previously demonstrated that open reading frame (ORF) 50 and ORF 57 encode transcriptional regulating genes in herpesvirus saimiri. ORF 50, a homolog of Epstein-Barr virus R protein, is a sequence-specific transactivator, whereas ORF 57 acts posttranscriptionally. In this report, we demonstrate that the ORF 57 gene is regulated by the ORF 50a gene product. We show that the ORF 57 gene is expressed at basal levels early in the virus replication cycle and that thereafter it is transactivated by the ORF 50a gene product, due to an increase in RNA levels. As it has been shown that the ORF 57 gene product downregulates ORF 50a due to the presence of its intron, these combined observations identify a feedback mechanism modulating gene expression in herpesvirus saimiri, whereby ORF 50a transcription is downregulated by the ORF 57 gene product, a gene which it specifically transactivates. Furthermore, we propose that the intron-containing ORF 57 gene downregulates itself by the same mechanism as that for ORF 50a, as both genes are downregulated at similar times during the replication cycle.

FIG. 1

Analysis of the ORF 57 promoter. OMK cell monolayers were transfected with 2 μg of pORF57CAT1 by using DOTAP (Boehringer Mannheim), as directed by the manufacturer; cells containing pORF57CAT1 subsequently either remained uninfected or were superinfected with 50 PFU of HVS (strain A11) per cell at 24 h posttransfection. Cells were harvested at 48 h posttransfection, and extracts were assayed for CAT activity. Products from these assays were separated by thin-layer chromotography and detected by autoradiography. Percentages of acetylation were calculated by the scintillation counting of the appropriate regions of the chromatography plate and are shown in graphic format.

FIG. 2

Mapping the gene responsible for ORF 57 transactivation. OMK cell monolayers were transfected with 2 μg of pORF57CAT1 and a series of plasmids which contained the entire L-DNA region of HVS (strain A11). Cells were harvested at 48 h posttransfection, and extracts were assayed for CAT activity. Products from these assays were separated by thin-layer chromotography and detected by autoradiography. Percentages of acetylation were calculated by the scintillation counting of the appropriate regions of the chromatography plate; error bars indicate the variations among three replicate assays.

FIG. 3

ORF 57 is transactivated by the ORF 50a gene product. (a) Schematic representation of the location of ORF 50a and ORF 50b within the L-DNA of HVS. (b) OMK cell monolayers were transfected with 2 μg of pORF57CAT1 in the absence and presence of either pORF50a or pAWHincII, which encodes ORF 50a or ORF 50b, respectively. Cells were harvested at 48 h posttransfection, and extracts were assayed for CAT activity. Products from these assays were separated by thin-layer chromotography and detected by autoradiography. Percentages of acetylation were calculated by the scintillation counting of the appropriate regions of the chromatography plate; error bars indicate the variations among three replicate assays.

FIG. 4

Immunofluorescence analysis of ORF 57 transactivation. OMK cells were transfected with 2 μg of pUCORF57 in the absence (a) and presence (b) of pORF50a, incubated with an ORF 57-specific antiserum, and then stained with fluorescein-conjugated anti-mouse immunoglobulin.

FIG. 5

RNA analysis of ORF 57 transactivation. Total RNA was isolated from OMK cells (lane 1), OMK cells transfected with pORF57CAT1 (lane 2), and OMK cells cotransfected with pORF57CAT1 and pORF50a (lane 3) at 24 hpi and hybridized with a ³²P-labelled primer homologous to the CAT coding region. Primer extension was performed, and the primer extension products were run on a 6% acrylamide–7 M urea gel and visualized by exposure to X-ray film. Mwt, molecular weight in thousands.

FIG. 6

ORF 57 transactivation requires the ORF 50 response elements contained within its promoter. (a) An ORF 57 promoter was generated by PCR amplification which deleted the putative ORF 50 response elements (RE); this fragment was ligated upstream of the CAT coding region to derive pORF57CAT2. (b) OMK cell monolayers were transfected with 2 μg of pORF57CAT2 in the absence and presence of pORF50a. Cells were harvested at 48 h posttransfection, and extracts were assayed for CAT activity. Products from these assays were separated by thin-layer chromotography and detected by autoradiography. Percentages of acetylation were calculated by the scintillation counting of the appropriate regions of the chromatography plate; error bars indicate the variations among three replicate assays.

FIG. 7

Gel retardation analysis. To locate the ORF 50 response elements contained within the ORF 57 gene promoter, gel retardation assays were performed on oligonucleotides mapping to this region. (a) Set 1: untransfected cells (lane 1) and pORF50a-transfected cells (lane 2); set 2: untransfected cells (lane 3) and pORF50a-transfected cells (lane 4). (b) Gel retardation experiments were repeated in the presence of increased amounts of unlabelled set 1 oligonucleotides. The retarded complexes were separated on a 5% polyacrylamide gel, run in 1% Tris-borate-EDTA buffer, and detected by autoradiography.

FIG. 8

Northern blot analysis of the expression of ORF 57 and ORF 50 genes. RNA was isolated from infected cells at intervals of 3 h up to 24 hpi and then at intervals of 6 h until 48 hpi, separated by electrophoresis on a 1% denaturing formaldehyde agarose gel, blotted onto a nylon membrane, and hybridized with radiolabelled probes specific for ORF 57 (a) and ORF 50 (b). Also shown are results of determination of RNA loading by hybridization with a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe (c).

FIG. 9

Schematic representation of the role and interactions of the ORF 57 and ORF 50 genes which regulate gene expression in the HVS replication cycle. DE, delayed early; REs, response elements.