The Immediate Early Genes of Human Cytomegalovirus Require Only Proximal Promoter Elements to Upregulate Expression of Interleukin-ld John W. Crump, Lois J. Geist, Philip E. Auron, Andrew C. Webb, Mark F. Stinski, and Gary W. Hunninghake Pulmonary Disease Division, Departments of Internal Medicine, Veterans Affairs, and Microbiology, University of Iowa College of Medicine, Iowa City, Iowa; Massachusetts General Hospital, Lovett Research Facility/Martin Laboratories, Charlestown, Massachusetts; and Department of Biological Sciences, Wellesley College, Wellesley, Massachusetts

Human cytomegalovirus (HCMV) can infect monocytes and macrophages. The immediate early one (lEI) gene product of HCMV positively regulates its own expression, as well as the expression of the interleukin1[3 (IL-l) gene. This study describes the IL-l promoter proximal region required for upregulation of IL-l gene expression by the HCMV lEI or lEI plus IE2 gene products. An IL-l chloramphenicol acetyltransferase (CAT) construct containing the IL-l genomic upstream sequence from position -1097 to +14 and four additional IL-ICAT plasmids containing progressive deletions of the -1097 to -131 sequence were used to evaluate the effect of the HCMV IE gene products on IL-l gene expression. IL-ICATplasmids were transfected into a monocytic cell line, THP-l, with plasmids containing either the IE promoter-regulatory region upstream of the bona fide lEI (pIEl), IE2 (pIE2), or lEI + 2 genes (pIE1+ 2) or a control plasmid containing the IE promoter-regulatory region alone (pLink760). In the presence ofpIEl+2, there was an approximate 15-fold increase in CAT activity compared with-the control, pLink760, in cells with CAT plasmids containing the -1097 to +14 IL-l sequence. Plasmids with progressive deletions of this sequence, including the plasmid containing the shortest upstream segment (-131 to +14) also had an approximate 15-fold increase in CATactivity. The upregulation of IL-l expression was mediated, primarily, by lEI and not by IE2. This effectwas promoter specific because an IL-ICATplasmid with a complete deletion of the proximal promoter elements (-234 to +146) did not respond to the HCMV IE gene products. These studies indicate that HCMV IE gene products require only proximal promoter elements from -131 to +14 to upregulate IL-l gene expression.

Human cytomegalovirus (HCMV) is present in a latent state in most healthy adults. Reactivation with potential clinical infection by HCMV may occur if the host becomes immunocompromised (reviewed in references 1 and 2). An active HCMV infection is associated with the sequential expression of the viral immediate early (IE), early, and late genes (3, 4). HCMV IE gene products modulate their own expression, as well as that of both the early and late HCMV genes (reviewed in references 5 and 6). IE genes also modulate the expression of other viralgenes, including those of the hu-

(Received in original form October 11, 1991 and in revisedform December 16, 1991) Address correspondence to: Lois 1. Geist, M.D., Assistant Professor, Pulmonary Disease Division, C33, GR, University of Iowa College of Medicine, Iowa City, IA 52242. Abbreviations: chloramphenicol acetyltransferase, CAT; human cytomegalovirus, HCMV; immediate early, IE; interleukin-lji, IL-l; lipopolysaccharide, LPS. Am. J. Respir. Cell Mol. BioI. Vol. 6. pp. 674-677, 1992

man immunodeficiency virus (7) and adenovirus (8-10). The HCMV IE gene products have been shown to upregulate the interleukin-l[3 (IL-l) gene in infected monocytes as well as in transient transfection assays (11, 12). This latter observation may have clinical relevance because HCMV IE genes are expressed in the monocytes that secrete IL-l (13-15). These observations suggest that HCMV IE gene products may have a role in affecting host inflammatory responses. The mechanisms by which the HCMV IE gene products regulate viral or cellular gene expression are not known. These viral gene products may affect the inflammatory processes, in part, by an interaction of the IE gene products with cellular DNA or regulatory proteins. To identify specific areas in the IL-l promoter-regulatory region that may interact with the HCMV IE gene products, we cotransfected IL-l chloramphenicol acetyltransferase (CAT) constructs with the HCMV IE genes into the monocytic cell line, THP-l. IL-ICAT plasmids containing progressive deletions of a portion of the IL-l gene containing the transcription start site and approximately 1 kb of upstream sequence were used. CAT activity was used to assess the effects of the HCMV IE gene products on expression of the IL-l gene.

Crump, Geist, Auron et al.: Human Cytomegalovirus and Interleukin-Id

Materials and Methods Cell Culture THP-1 cells, a myelomonocytic cell line obtained from American Type Culture Collection (Rockville, MD), were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 4 mM glutamine, and 80 p,g/ml gentamicin. Cells were transfected during the log phase of their growth. Plasmid Constructs Constructions of all IE plasmids have been described previously and shown to express the bona fide viral gene products (8, 16-18). (See Figure 1, upper panel, for a schematic diagram of these plasmids and Malone and co-workers [17] for details.) The parental IL-ICAT plasmid (PT) used in these studies consists of the human IL-lt3 gene from position -1097 to +14 derived from the BDC-454 clone, described by Clark and colleagues, inserted upstream of the gene coding for CAT in plasmid pCAT3M (16, 19, 20). The deletion plasmids DT, NT, and HT are identical to PT with the exception that the upstream genomic sequence has been progressively truncated such that the upstream ends correspond to positions -313, -234, and -131, respectively. The 3'terminus in each case is located at +14. Clone AT,unlike the other clones, contains sequences that correspond to - 512 to +14, as well as an additional 40 nucleotides derived from the IL-1 genomic sequence in the region -2588 to -2548. Plasmid PX~N is derived from PT. The 5' terminus is at -1097 with a deletion of the region between -234 to + 146. These plasmids are schematically represented in Figure 1, lower panel. Transfection Transfections were performed using the DEAE dextran method as described previously (12, 21). Briefly, the cells were washed and resuspended in serum-free medium at a concentration of 1 x lOS cells/ml in 10 ml of DEAE dextran, 250 p,g/ml, for 1 h. The cells were then washed and resuspended in medium containing 10% fetal bovine serum. Stimulation was carried out by adding lipopolysaccharide (LPS) 24 h after transfection. The cells were harvested after an additional 24 h in culture. CAT Assay CAT assays were performed by the method of Gorman and associates (22). Ascending thin-layer chromatography was used to separate the acetylated derivatives of chloramphenicol using a chloroform:methanol solvent system (95:5). Autoradiograms were developed after overnight exposure. CAT activity was quantitated using a TLC scanner (Radiomatic Instruments, Tampa, FL).

Results To evaluate the effectsof the HCMV IE gene products on IL1CATexpression, the IE1+2 expression plasmid, pIE1+2, or a control plasmid, pLink760, was transfected into THP-1 cells with the parental IL-1CAT plasmid (PT) or one of the IL-1CAT deletion plasmids (Figure 1). The THP-l cells were stimulated with LPS (100 J,tglml) for 24 h and then assayed for CAT activity. As previously shown (l2), PT in the presence of pIEl + 2 increased IL-l promoter activity 15-fold

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Figure 1. Upper panel: Schematic representation of the human cytomegalovirus (HCMV) immediate early (IE) plasmid constructs. Lower panel: Schematic representation of the interleukin-l (lL-l) chloramphenicol acetyltransferase (CAT) plasmid constructs. The AT construct contains a 40-bp segment derived from the region -2588 to -2548 of the upstream IL-l genomic sequence. In the plasmid, this segment is located upstream of -512 (hatched region).

(Figure 2, upper panel). A similar degree of enhancement of CAT activity was seen with each of the IL-1CAT deletion plasmids that represent a progressive shortening of the IL-1 upstream genomic sequence, to 131 bp (Figure 2, upper and middle panels). In contrast, the PXdN plasmid, containing an internal deletion from -234 to +146, showed no measurable increase in CAT activity in the presence of the HCMV IE1+2 gene products (Figure 2, lower panel). These data suggest that the proximal elements of the IL-1 promoter region are critical for upregulation by the HCMV IE gene products. To determine whether differences in IL-ICAT activity with the IL-1CAT deletion plasmids could be detected by using different amounts of LPS, THP-1 cells were cotransfected with the HCMV IE1+2 plasmid and the IL-1CAT plasmids. After 24 h, the cells were either left unstimulated or were stimulated with LPS at 10 or 100 p,g/ml. The cells were then harvested after an additional 24 h in culture. In the presence of the HCMV IE1+2 gene products, LPS exerted a dose-response effect on IL-1 promoter activity as shown by increased CAT activity (Figure 3). Each of the IL1CAT plasmids, all of which contained the -131 to +14 sequence, showed significant and similar baseline promoter activity in the presence of only the HCMV lEI + 2 gene products. Addition of LPS at 10 p,glml resulted in a 5- to 10-fold increase in activity over control and a 15- to 20-fold increase in activity with LPS at a concentration of 100 J,tg/ml. With each concentration of LPS, there were no significant differences in amounts of CATactivity among the various IL-1CAT plasmids.

676

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 6 1992

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Figure 3. Dose-response effect ofLPS on the expression of various IL-ICAT deletion plasmids in the presence of IE1+2. THP-1 cells were transfected, using the DEAE dextran (250 ILg/ml) transfection method (12, 21), with the full-length IL-lCAT plasmid or various IL-ICAT deletion plasm ids. Twenty four hours after transfection, the cells either remained unstimulated (black bars) or were stimulated with LPS at a concentration of either 10 p.g/ml (closed hatched bars) or 100 p.g/ml (stippled bars). CAT assays were performed as described in MATERIALS AND METHODS. The IL-1CAT deletion plasmids are indicated on the abscissa. IL-1CAT activity is expressed on the ordinate as percent conversion of ['4C]chloramphenicol to its acetylated derivatives. The figure represents data from three experiments, expressed as mean ± SEM.

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To demonstrate that the upregulation of the IL-l promoter remained specific to the HCMV lEI gene product, THP-l cells were co-transfected with the shortest IL-l upstream genomic sequence (-131 to +14, HT) and one of the HCMV plasmids (pLink, pIEl, pIE2, and pIEl+2). The presence of the HCMV lEI gene product alone increased HT CATactivity, whereas the IE2 gene product alone had no effect. As discussed above, pIEI +2 also had a significant effecton the IL-l promoter activity (Figure 4).

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Figure 2. Effects of progressive deletions of the IL-1 promoterregulatory region on the capacity of the HCMV IE gene products to upregulate expression of the IL-l promoter. THP-l cells were cotransfected, using the DEAE dextran (250 ILg/ml) transfection method (12, 21) with the control plasmid (pLink 760) (open hatched bars) or the pIE1+2 plasmid (closed hatched bars) and one of the IL-ICAT deletion plasmids that progressively shortens the IL-l promoter-regulatory region from -1097 to -131. Twenty four hours after transfection, the cells were stimulated with lipopolysac-

charide (LPS) (100 p.g/ml)for an additional 24 h. CAT assays were performed as described in MATERIALS AND METHODS. The ILlCAT deletion plasmids are indicated on the abscissa. IL-ICAT activity is expressed on the ordinate as percent conversion of [i4C]chloramphenicol to its acetylated derivatives. Upper panel: Representative experiment showing that progressive deletion of the IL-l promoter-regulatory region from -1097 to -131 does not alter upregulation of the IL-l promoter by HCMV IE gene products. Middle panel: Mean + SEM of three experiments illustrated in the upper panel. Lower panel: Mean + SEM of three experiments showing that the proximal promoter elements of IL-l are required for upregulation by HCMV IE gene products.

Crump, Geist, Auron et al.: Human Cytomegalovirus and Interleukin-1{3

677

References

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Figure 4. A representative experiment demonstrating the effect of the individual HCMV IE gene products on expression of the proximal promoter of IL-1 deletion plasmid (HT). THP-1 cells were transfected using the DEAE dextran (250 tLg/ml) transfection method (12, 21) with the IL-1CAT plasmid HT (-131 to +14) and either pLink 760 (control), pIE1, pIE2, or pIE1+2. The cells remained unstimulated or, 24 h after transfection, were stimulated with LPS (10 tLg/ml) for an additional 24 h. CAT assays were performed as described in MATERIALS AND METHODS.

length IL-1 promoter (12) and demonstrate that the region essential for lEI to activate the IL-1 promoter is localized to between -131 and +14. We found that longer IL-1 promoter sequences, up to -1097, were no more effective at mediating both the response to the HCMV IE gene products and the response to LPS activation than were the proximal promoter elements of the IL-1 gene. This effect was specific because deletion of the promoter proximal element (-131 to +14) resulted in no upregulation of CAT activity in the presence of the IE gene products. Thus, although many transacting factors increase transcription via upstream enhancer elements, these studies clearly show that the HCMV lEI gene product increases transcription of the IL-1 gene via an effect on proximal promoter elements. Although the mechanisms by which LPS stimulates IL-1 gene expression are not well understood, it is likely that LPS induces factors within the cell nucleus that interact with the IL-1 gene (23). This effect is supported by our previous findings that the amount of IL-1 promoter activity is related to the amount of lEI plasmid transfected into the cells (12). The region between -131 and + 14 contains consensus sequences for several previously characterized transcription factors as well as a binding site for a monocyte-specific factor (19, 24). It is not known at this time whether this site is also critical for modulation of IL-1 gene expression by lEI or LPS. Further work is necessary to determine how the IE gene product of HCMV effects IL-1 gene expression. Cherrington and Mocarski (25) suggested that the lEI gene product may upregulate its own promoter via an 18-bp repeat that contains an NF-KB motif. The IL-1 gene sequence between -131 and +14, however, does not contain an NF-KB site. Therefore, the lEI upregulation of the IL-1 promoter containing only 131 bp upstream from the transcription initiation site occurs in the absence of the NF-KB motif. It is possible that the lEI protein of HCMV interacts with multiple components of the transcription complex. Acknowledgments: The writers wish to thank Drs. Burton D. Clark and Matthew J. Fenton for helpful input and discussion. This study was supported by VA Merit Review, NHLBI/NIH P/SCOR HL37121 (to Dr. Hunninghake), NIH/AI 13562 (to Dr. Stinski) AHA-Iowa Affiliate Fellowship (to Dr. Geist), NIH/AI 27850 (to Dr. Auron), and RR59 NIH/Clinical Research Centers grants.

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