Rapid Communication The Immediate Early Genes of Human Cytomegalovirus Upregulate Expression of the Cellular Genes myc and los Martha M. Monick, Lois J. Geist, Mark F. Stinski, and Gary W. Hunninghake Departments of Internal Medicine and Microbiology, University of Iowa College of Medicine, and Department of Veterans Affairs Medical Center, Iowa City, Iowa

Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (lEI and IE2) can alter the regulation of the cellular immediate early genes (c-fos and c-myc). Plasmid constructs containing the promoter-regulatory regions c-myc or c-fos upstream of the reporter gene, chloramphemicol acetyl transferase, were co-transfected into T cells (Jurkat cells), monocytes/macrophages (THP-I cells), or human fibroblast cells with plasmid constructs containing the promoter-regulatory region of the HCMV IE genes upstream of the bona fide lEI, IE2 or IE+2 genes; a plasmid that contained no IE coding region was used as a control. These studies show that both products of the HCMV IE genes markedly upregulated expression of the cellular c-fos and c-myc genes. The viral effects of individual proteins (lEI or IE2) were dependent both on the promoter-regulatory region of the cellular gene and the cell type. In all cells, the combination ofIEI and IE2 further upregulated both cellular genes, suggesting a synergistic effect of lEI with IE2. Both of the c-myc promoters (PI and P2) were upregulated by the HCMV IE gene products. lEI and IE2 also upregulated the cells' endogenous c-myc and c-fos genes, as determined by amounts of the respective mRNAs. These studies show that HCMV can markedly alter cellular IE gene expression and that the effects of HCMV lEI and IE2 proteins are dependent both on the promoter-regulatory region of the cellular gene and the type of cell in which the interaction occurs.

Human cytomegalovirus (HCMV) is a frequent cause of lung disease in immunocompromised patients (1). During active infection, there is a sequential expression of the immediate early (IE), early, and late HCMV genes (2). HCMV can also cause a latent infection that is characterized by expression of only the IE genes. The IE genes code for proteins that are known to regulate their own production as well as the expression of early and late viral genes (3-9). HCMV infection is associated with increased cellular DNA synthesis and cell proliferation (10-12). Two cellderived gene products that play an important role in cell proliferation and differentiation are the proto-oncogenes c-fos and c-myc (13-19). Boldogh and colleagues recently reported that HCMV infection can increase the levels of both (Received in original form April 24. 1992 and in revisedform May 29. 1992) Address correspondence to: Gary W. Hunninghake, M.D., Pulmonary Disease Division, C33, GH, Department of Internal Medicine, University of Iowa, College of Medicine, Iowa City, IA 52242. Abbreviations: human cytomegalovirus, HCMV; immediate early, IE; lipopolysaccharide, LPS; phorbol myristate acetate, PMA; thin-layer chromatography, TLC. \ Am. J. Respir. CeU Mol. BioI. Vol. 7. pp. 251-256, 1992

c-myc and c-fos steady-state RNA in human embryonic lung fibroblasts (20). The increases in mRNAs were due to enhanced transcription (21). Since this increase in transcription was detectable with either UV-inactivated virus or infectious virus in the presence of an inhibitor of de novo protein synthesis, but not with proteinase-treated virus, they postulated that a virion-associated factor was responsible for the increase in transcription. We investigated whether the products of the HCMV IE genes, which are expressed during both active and latent infections, could upregulate c-myc and c-fos. We found that products of the HCMV IE genes markedly increase endogenous c-myc and c-fos RNA. In addition, using transient transfection assays, we show that HCMV IE gene products markedly upregulate the c-myc and c-fos promoters in three different cell lines.

Materials and Methods Reagents Lipopolysaccharide (LPS; Escherichia coli 026:B6), phorbol myristate acetate (PMA), guanidine isothiocyanate, and cesium chloride were obtained from Sigma Chemical Co.

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(St. Louis, MO). DEAE dextran and acetyl coenzyme A were obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Gene Screen Plus, P2P]CTP, and 14C-labeled chloramphenicol were obtained from DuPont, NEN Research Products (Boston, MA).

Cat Assays

Tissue Culture Jurkatcells (a T-cell line) were maintained in suspension culture in RPMI 1640 medium containing 10% fetal bovine serum (Sigma), 2 mM L-glutamine, and 80 /Lg/mlgentamycin (complete medium) at 37° C in a 5% CO 2 atmosphere. THP-1 cells, a myelomonocytic cell line, were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in suspension culture in the same medium, as described above. CCD-18LU cells, a fibroblast-like cell line derived from human lung tissue (ATCC), were maintained as adherent cells in the same medium.

Cell extracts were prepared and assayed as previously described (25,27). One hundred microliters of cell extract was incubated with 0.1 /LCi of [l4C]chloramphenicol and 1 mM acetyl coenzyme A for 90 min at 37° C. The percentage of [l4C]chloramphenicol that was converted to its acetylated derivatives was determined using ascending thin-layer chromatography (TLC) with a chloroform:methanol (95:5) solvent. Radioactivity was quantitated using an automated RTLC scanner (Radiomatic Instruments, Tampa, FL). Data was expressed as fold increase, compared to controls (pLink). All CAT assays were performed within the linear range of the assay.

Plasmid Constructs Construction of all IE plasmids has been described previously (7, 9, 22). The plasmid, pLink760 (control) contains only the HCMV IE promoter-regulatory region and consists of a 760-bp Sau3A-I fragment cloned into the bacterial vector pATl53. The plasmid pIEl (PSVCC1) contains the HCMV IE promoter-regulatory region upstream of the bonafide lEI gene; it contains the 3.6-kb Clal restriction fragment which spans the lEI gene as well as the HCMV IE promoter-regulatory region cloned into the plasmid pSVOd. Plasmid plE2 (pCSdlAcc) contains the HCMV IE promoter-regulatory region upstream of the bonafide IE2 gene; it contains a BamHI to SalI fragment containing the IE2 coding region and an ACCI to ACCI deletion of exon 4 of the IE1 gene. Finally, plEI + 2 (pSVCS) contains the IE promoter-regulatory region upstream of both the lEI and IE2 genes. The mycCAT plasmid was obtained from U. Siebenlist (NIAID, NIH, Bethesda, MD), and contains a 2.9-kb fragment, ending at +348 (HindIII-PvuIl), of the human c-myc-derived promoter-regulatory region inserted in pSVO CAT. The fosCAT (FC4) plasmid was obtained from W. Lamph (Salk Institute, San Diego, CA) and contains a 404-bp fragment of the promoter-regulatory region of the human c-fos gene inserted into pSV2CAT (23). Three mycCAT deletion constructs were obtained from M. Lipp (lnstitut fur Biochemie der Universitat Munchen, Munich, Germany). mycCAT-P contains the sequence -511 to + 348 from the c-myc promoter-regulatory region inserted upstream of CAT in pUC12. mycCAT-P~P1 has a deletion from -263 to -95 in the mycCAT-P plasmid. mycCATP~P2 has a deletion from -95 to +348 in the mycCAT-P plasmid (24). Transfections Jurkat cells, THP-1 cells, and CCD-18LU cells were all transfected using the DEAE-dextran method as previously described (25, 26). Briefly, the cells were washed and then placed in 10 ml (10' Jurkat or THP-1 cells or a confluent 100-mm plate of CCD-18LU cells) of serum-free RPMI 1640 medium with 250 /Lglml DEAE dextran, 50 mM Tris, and 1 /Lglml of each DNA plasmid to be transfected. The cells were incubated for 1 h at 37° C and then washed and resuspended in RPMI-1640 medium with 10% fetal bovine serum. After an additional 24 h in culture, some cells were

stimulated with appropriate amounts of PMA or LPS. At 3, 6, or 24 h after stimulation, the cells were harvested and used either for CAT assays or as sources of RNA.

Dot Blot Analysis Whole-cell RNA was isolated as previously described (28). Briefly, cells were lysed with guanidine isothiocyanate, layered over a cesium chloride gradient, and spun overnight at 30,000 rpm. The RNA was quantitated spectrophotometrically and used in a dot blot assay by applying serial 2-fold dilutions of whole-cell RNA to a nylon membrane (Gene Screen Plus). The first sample of each series contained 10 /Lg of RNA. Probes used to detect c-fos and c-myc RNA were labeled with 32p by nick translation prior to hybridization at 42° C. Autoradiograms were quantified using laser densitometry. The c-myc probe (JN1B) was obtained from R. Kaufman (29). It contains a 1.4-kb segment (Cial to EcoRI) of the human c-myc gene which includes Exon 3. The c-fos probe (T7fos) was obtained from W. Lamph (30) and contains a full-length c-fos cDNA. On Northern analysis, the c-fos probe identified a single transcript at 2.2 kb and the c-myc probe identified a single transcript at 2.4 kb (data not shown). Neither probe reacted with ribosomal RNA.

Results Effect of HCMV IE Gene Products on Expression of myc To evaluate the effects of HCMV IE gene products on myc expression, Jurkat, THP-1, and human fibroblast cells were co-transfected with the mycCAT plasmid and one of the HCMV IE plasmids (pLink, p1E1, p1E2, or pIEH2). Jurkat cells were stimulated with PMA (10 nglml) and THP-1 cells were stimulated with LPS (10 /Lglml) 24 h after transfection; for both cell types, control cells remained unstimulated. The CCD-18LU cells all remained unstimulated. The HCMV IE genes are not expressed in unstimulated Jurkat cells or THP-1 cells but are expressed in stimulated cells (25). In contrast, the HCMV IE genes are expressed in unstimulated fibroblasts (5, 31-33) and stimulation of the cells does not increase the expression of these genes. PMA is a much more effective stimulus compared to LPS for the expression of the HCMV IE genes in Jurkat cells, whereas LPS is a more effective stimulus in THP-1 cells (data not shown). All groups of cells were harvested 48 h after transfection to evaluate levels of CAT activity. In Jurkat cells stimulated with PMA, the presence of the pLink or pIEl plasmids had no effect on mycCAT activity

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(Figure lA). The HCMV IE2 gene product increased CAT expression about 90-fold compared to the control (pLink) plasmid. When the plasmid containing both the IEI and IE2 genes was present, there was approximately a 420-fold increase in CAT activity compared to the control (pLink) plasmid. We also evaluated whether HCMV IE gene products could interact with either the PI and P2 promoters of the c-myc gene, using the plasmids mycCAT-P (which contains both the PI and P2 promoters), myc CAT-PdPI (containing only the P2 promoter), and mycCAT-PdP2 (containing only the PI promoter) (Figure 2). Each of these three plasmids was co-transfected into Jurkat cells with one of the HCMV IE plasmids, pLink, pIEI, pIE2, and pIEH2. All cells were stimulated with PMA (10 ng/rnl). The HCMV plasmid pIE2

600 A: Jurkat Cells • PMA

500 Q)

The immediate early genes of human cytomegalovirus upregulate expression of the cellular genes myc and fos.

Human cytomegalovirus (HCMV) is an important pathogen of the lung. We determined whether the HCMV immediate early genes (IE1 and IE2) can alter the re...
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