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. 1999 Nov;73(11):9039-52.
doi: 10.1128/JVI.73.11.9039-9052.1999.

A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer

C A Lundquist  1 , J L Meier, M F Stinski
Affiliations

A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer

C A Lundquist et al. J Virol. 1999 Nov.

Abstract

The region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region. DNase I protection analysis with human cell nuclear extracts demonstrated multiple protein binding sites in this region of the viral genome (P. Ghazal, H. Lubon, C. Reynolds-Kohler, L. Hennighausen, and J. A. Nelson, Virology 174:18-25, 1990). However, the function of this region in the context of the viral genome is not known. In wild-type human CMV-infected human fibroblasts, cells permissive for viral replication, there is little to no transcription from UL127. We determined that the unique region prevented transcription from the UL127 promoter but had no effect on the divergent MIE promoter. In transient-transfection assays, the basal level of expression from the UL127 promoter increased significantly when the wild-type unique sequences were mutated. In recombinant viruses with similar mutations in the unique region, expression from the UL127 promoter occurred only after de novo viral protein synthesis, typical of an early viral promoter. A 111-bp deletion-substitution of the unique sequence caused approximately a 20-fold increase in the steady-state level of RNA from the UL127 promoter and a 245-fold increase in the expression of a downstream indicator gene. This viral negative regulatory region was also mutated at approximately 50-bp regions proximal and distal to the UL127 promoter. Although some repressive effects were detected in the distal region, mutations of the region proximal to the UL127 promoter had the most significant effects on transcription. Within the proximal and distal regions, there are potential cis sites for known eucaryotic transcriptional repressor proteins. This region may also bind unknown viral proteins. We propose that the unique region upstream of the UL127 promoter and the MIE enhancer negatively regulates the expression from the UL127 promoter in permissive human fibroblast cells. This region may be a regulatory boundary preventing the effects of the very strong MIE enhancer on this promoter.

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Figures

FIG. 1
FIG. 1
Northern blotting of viral transcripts from the unique region and the UL127 gene at various times after infection. (A) Diagram of the unique long (UL) and unique short (US) components of the human CMV (HCMV) genome flanked by terminal repeat (TR) or inverted repeat (IR) sequences containing an a sequence. An EcoRI restriction endonuclease map of the viral genome and the EcoRI region of the wild-type (WT) virus and recombinant virus RdlMSVgpt(r1) is diagramed. Mock, IE, early, and late total cell RNAs were isolated and subjected to Northern blot hybridization with either a 32P-labeled bidirectional probe (B) or a 32P-labeled unidirectional probe (C). To determine the sensitivity of this method, RNA was synthesized in vitro from the UL127 gene template and analyzed. Standard molecular size markers are indicated in kilobases. SV40, simian virus 40.
FIG. 1
FIG. 1
Northern blotting of viral transcripts from the unique region and the UL127 gene at various times after infection. (A) Diagram of the unique long (UL) and unique short (US) components of the human CMV (HCMV) genome flanked by terminal repeat (TR) or inverted repeat (IR) sequences containing an a sequence. An EcoRI restriction endonuclease map of the viral genome and the EcoRI region of the wild-type (WT) virus and recombinant virus RdlMSVgpt(r1) is diagramed. Mock, IE, early, and late total cell RNAs were isolated and subjected to Northern blot hybridization with either a 32P-labeled bidirectional probe (B) or a 32P-labeled unidirectional probe (C). To determine the sensitivity of this method, RNA was synthesized in vitro from the UL127 gene template and analyzed. Standard molecular size markers are indicated in kilobases. SV40, simian virus 40.
FIG. 2
FIG. 2
Relative LUX activity from the UL127 promoter in transiently transfected HFFs. CAT or LUX activity per microgram of protein was measured from the MIE promoter or the divergent UL127 promoter, respectively, as described in Materials and Methods. The cis-acting transcription factor binding sites in repeat and nonrepeat sequences have been described elsewhere (52, 79, 80, 82, 83). Plasmids containing wild-type viral DNA sequence, deleted DNA sequence, or substituted nonregulatory stuffer DNA sequence are indicated relative to the MIE promoter transcription start site. The approximate positions of the mutations relative to the putative UL127 transcription start are also indicated. CAT activity was similar for all expression plasmids as described in the text.
FIG. 3
FIG. 3
Recombinant viruses with wild-type (wt or WT) or mutant DNA sequences upstream of the UL127 promoter. All recombinant viruses containing the CAT gene were derived from RdlMSVgpt(r1) by selection against the gpt gene product as described in Materials and Methods. (A) Diagram of the recombinant viruses with the MIE promoter and the downstream IE1 gene and the divergent UL127 promoter and the downstream CAT gene. Both promoters have identical TATA boxes. Sequences upstream of the UL127 promoter with either wild type or deleted and replaced with nonregulatory DNA sequence of either 116, 121, or 134 bp. The DNA fragment containing the UL127 promoter is deleted in recombinant virus RdlMCATTheta. The origins of probes for detection of wild-type and 116-, 121-, or 134-bp heterologous DNAs are designated. The predicted sizes of DNA fragments after EcoRI and NdeI restriction endonuclease digestion of recombinant viral DNAs are indicated in base pairs. HCMV, human CMV. SV40, simian virus 40. (B) Southern blot hybridization of wild-type (WT) Towne strain DNA and recombinant virus DNA digested with restriction endonucleases EcoRI and NdeI. 32P-labeled probes of wild-type and 116-, 121-, and 134-bp heterologous DNA sequences are indicated. Lanes: 1 to 6, viral DNAs from human CMV Towne strain and RdlMCATWT, -Theta, -116, -121, and -134, respectively; 1 to 4 (at right), viral DNA from human CMV Towne strain and RdlMCATWT, -Theta, and -134, respectively. Standard molecular size markers are indicated in base pairs.
FIG. 4
FIG. 4
Transcription from the MIE promoter or the UL127 promoter at various times after infection with recombinant viruses. Cytoplasmic RNA was isolated from either mock-infected or infected cells treated with CH or PAA or untreated for IE, early, or late times after infection, respectively. (A) RNase protection assay of IE1 or UL127-CAT RNA harvested at 6 h p.i. from nontreated or CH-treated cells. Lanes: 1, standard molecular weight markers (std) (in thousands); 2, mock-infected cell RNA; 3 to 7 and 8 to 12, infected cell RNA from RdlMCATWT, -116, -121, -134, and -Theta, respectively. A map of the riboprobe and the protected fragment is shown. nt, nucleotide; AS, antisense riboprobe. (B) RNase protection assay of IE1 or UL127-CAT RNA harvested at 24 or 48 h p.i. from nontreated or PAA-treated cells. Lanes: 1, IE1 probe without RNase; 2, standard molecular weight markers (std) (in thousands); 3, CAT-UL127 probe without RNase; 4, mock-infected cell RNA; 5 to 7, 8 to 10, and 11 to 13, infected cell RNA from RdlMCATWT, -121, and -Theta, respectively. CAT-UL127 and IE1 RNase-protected RNA are indicated.
FIG. 5
FIG. 5
Expression of the CAT gene downstream of the UL127 promoter at various times after infection with recombinant viruses containing wild-type (WT), promoterless, or substituted heterologous DNA sequence upstream of the UL127 promoter. The recombinant viruses are as described for Fig. 3. CAT activity was determined as percent acetylation per microgram of protein-infected cell lysate as described in Materials and Methods.
FIG. 6
FIG. 6
Recombinant viruses with approximately 50-bp deletions upstream of the UL127 promoter. All recombinant viruses containing the CAT gene were derived from RdlMSVgpt(r1) by selection against the gpt gene product as described in Materials and Methods. (A) Diagram of the recombinant viruses with the MIE promoter and the downstream IE1 gene and the divergent UL127 promoter and the downstream CAT gene. Approximately 50-bp deletions between −694 and −640 or −640 and −583 are indicated. The origin of a probe for detection of wild-type (WT or wt) DNA is designated. The predicted sizes of DNA fragments after EcoRI and NdeI restriction endonuclease digestion of recombinant viral DNAs are indicated in base pairs. HCMV, human CMV. SV40, simian virus 40. (B) Southern blot hybridization of wild-type Towne strain DNA and recombinant viral DNAs digested with restriction endonucleases EcoRI and NdeI and fractionated by agarose gel electrophoresis as described in Materials and Methods. Lanes: 1, human CMV Towne strain; 2, RdlMCATWT; 3, RdlMCAT694-640A; 4, RdlMCAT694-640B; 5, RdlMCAT640-583A; 6, RdlMCAT640-583B; 7, RdlMCAT640-583C. Numbers at left indicate molecular sizes in kilobases.
FIG. 7
FIG. 7
Effect of either the proximal or distal deletions of approximately 50 bp on transcription from the UL127 promoter at early times after infection. Infected cell RNA was isolated at 6 h p.i. and analyzed as described in the legend to Fig. 4. Lanes: 1, standard molecular weight markers (std) (in thousands); 2, mock-infected cell RNA; 3, infected cell RNA from RdlMCATWT; 4 to 7, infected cell RNA from RdlMCAT694-640A, -B, -C, and -D, respectively; 8 and 9, infected cell RNA from RdlMCAT640-583A and -C, respectively; 10, IE1 probe not treated with RNase; 11, CAT-UL127 probe not treated with RNase; CAT-UL127 and IE1 RNase-protected RNAs are indicated.
FIG. 8
FIG. 8
Expression of the CAT gene downstream of the UL127 promoter at various times after infection with recombinant viruses containing proximal or distal deletions of approximately 50 bp upstream of the UL127 promoter. The recombinant viruses are as described for Fig. 6. CAT activity was determined as percent acetylation per microgram of protein of infected cell lysate as described in Materials and Methods.
FIG. 9
FIG. 9
Diagram of the region between the UL127 promoter and the MIE enhancer demonstrating putative binding sites for winged-helix transcriptional repressor, NF-1, suHW, and the HPV silencing motif (PSM).
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