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. 1999 Apr;73(4):2825-31.
doi: 10.1128/JVI.73.4.2825-2831.1999.

Effects of human cytomegalovirus major immediate-early proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells

D M Lukac  1 , J C Alwine
Affiliations

Effects of human cytomegalovirus major immediate-early proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells

D M Lukac et al. J Virol. 1999 Apr.

Abstract

The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) encodes several MIE proteins (MIEPs) produced as a result of alternative splicing and polyadenylation of the primary transcript. Previously we demonstrated that the HCMV MIEPs expressed from the entire MIE gene could rescue the temperature-sensitive (ts) transcriptional defect in the ts13 cell line. This defect is caused by a ts mutation in TAFII250, the 250-kDa TATA binding protein-associated factor (TAF). These and other data suggested that the MIEPs perform a TAF-like function in complex with the basal transcription factor TFIID. In addition to the transcriptional defect, the ts mutation in ts13 cells results in a defect in cell cycle progression which ultimately leads to apoptosis. Since all of these defects can be rescued by wild-type TAFII250, we asked whether the MIEPs could rescue the cell cycle defect and/or affect the progression to apoptosis. We have found that the MIEPs, expressed from the entire MIE gene, do not rescue the cell cycle block in ts13 cells grown at the nonpermissive temperature. However, despite the maintenance of the cell cycle block, the ts13 cells which express the MIEPs are resistant to apoptosis. MIEP mutants, which have previously been shown to be defective in rescuing the ts transcriptional defect, maintained the ability to inhibit apoptosis. Hence, the MIEP functions which affect transcription appear to be separable from the functions which inhibit apoptosis. We discuss these data in the light of the HCMV life cycle and the possibility that the MIEPs promote cellular transformation by a "hit-and-run" mechanism.

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Figures

FIG. 1
FIG. 1
Diagram of the MIE gene of HCMV showing alternatively spliced and polyadenylated (PA) transcripts produced from its primary transcript. The transcripts encoding the 72-, 86-, and 55-kDa MIEPs (IEP72, IEP86, and IEP55) are shown. The various exons are numbered; open reading frames (ORFs) within exons are shaded. The IE1 region is composed of exons 1 to 4, and the IE2 region consists of exon 7. Note that IEP55 differs from IEP86 by having an extra splice, resulting in the removal of an IEP86 unique region in exon 7. The IE2-derived ORFs flanking the UR are labeled as exons 5 and 6, specific to IEP55. In some diagrams of the MIE region the exon we have called 7 is numbered as exon 5.
FIG. 2
FIG. 2
Effect of MIEP expression on the block to cell cycle progression in ts13 cells. Cultures of ts13 cells were transfected with pXJ40E/B (control), pRL43a (encoding the MIEPs), or pCMV-hTAFII250 (encoding hTAFII250). In addition, the cells were cotransfected with plasmid pc-hCD4, which expresses CD4. The transfected cells were then subjected to FACS analysis for DNA content; only CD4-positive cells were gated so that the transfected cells would be preferentially analyzed (see Materials and Methods). Cell cycle modeling and quantitation were performed with ModFit software. The bars show the percentage of cells in the various phases of the cell cycle. The data show representative results from an experiment where samples were tested in triplicate. Similar results were obtained in five separate experiments. The first (solid) bar in each phase indicates control plasmid-transfected cells at 32°C, the second (lightly shaded) bar indicates control plasmid-transfected cells at 39°C, the third (darkly shaded) bar indicates MIEP-expressing cells at 39°C, and the fourth (open) bar indicates hTAFII250-expressing cells at 39°C. The error bars indicate standard deviations.
FIG. 3
FIG. 3
Effect of MIEP expression on apoptosis in ts13 cells grown at the nonpermissive temperature. Coverslips containing ts13 cells grown at 32°C were transfected with either pSVHwt (upper panels), which encodes the WT MIEPs, or pSVHm13 (lower panels), which encodes MIEPs which are defective in transcriptional activation (40). The cultures were incubated at 39°C for 24 to 28 h posttransfection and then fixed. The fixed coverslips were tested for (i) immunofluorescence detection of the MIEPs (left panel in each set) and (ii) immunofluorescence detection of apoptosis (by TUNEL assay; right panel in each set) (see Materials and Methods for details). Identical fields of cells are shown for MIEP detection (red) and apoptosis (yellow). For comparison, the arrows indicate the positions of MIEP-expressing cells in the fields.
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