Transcriptional regulation of the human cytomegalovirus US11 early gene

Abstract

The human cytomegalovirus (HCMV) US11 early gene encodes a protein involved in the down-regulation of major histocompatibility complex class I cell surface expression in HCMV-infected cells. Consequently, this gene is thought to play an important role in HCMV evasion of immune recognition. In this study, we examined the transcriptional regulation of US11 gene expression. Analysis of deletions within the US11 promoter suggests that two sequence elements are important for activation by the viral immediate-early (IE) proteins. Deletion of a CREB site located at -83 relative to the cap site resulted in a reduction in promoter activity to 50% of the wild-type level. Deletion of an additional ATF site immediately upstream of the TATA box resulted in abrogation of responsiveness to the IE proteins. To confirm the role of the CREB and ATF sites within the US11 promoter, mutagenesis of these two sites, both individually and in combination, was carried out. Results indicate that both the CREB element and the ATF site were required for full promoter activity, with the ATF site critical for US11 promoter activation. The loss of transcriptional activation correlated with a loss of cellular proteins binding to the mutated US11 promoter elements. In combination with the viral IE proteins, the HCMV tegument protein pp71 (UL82) was found to up-regulate the US11 promoter by six- to sevenfold in transient assays. These results suggest that pp71 may contribute to the activation of the US11 promoter at early times after infection. Up-regulation by pp71 required the presence of the CREB and ATF sites within the US11 promoter for full activation. The role of the ATF and CREB elements in regulating US11 gene expression during viral infection was then assessed. The US11 gene is not required for replication of HCMV in tissue culture. This property was exploited to generate US11 promoter mutants regulating expression of the endogenous US11 gene in the natural genomic context. We generated recombinant HCMV that contained the US11 promoter with mutations in either the CREB or ATF element or both regulating the expression of the endogenous US11 gene. Northern blot analysis of infected cell mRNA revealed that mutation of the CREB element reduced US11 mRNA expression to approximately 25% of that of the wild-type promoter, with identical kinetics of expression. Mutation of the ATF site alone reduced US11 mRNA levels to 6% of that of the wild-type promoter, with mRNA detectable only at 8 h after infection. Mutation of both the CREB and ATF elements in the US11 promoter reduced US11 gene expression to undetectable levels. These results demonstrate that the CREB and ATF sites cooperate to regulate the US11 promoter in HCMV-infected cells.

FIG. 1

Sequence of the US11 promoter. The TATA element is indicated in bold. Putative binding sites for the transcription factors ATF, CREB, and CF1 are boxed. In addition, two copies of a direct repeat element (DR1) as well as a palindromic sequence (P1) are indicated.

FIG. 2

(A) Schematic diagram of the US11 promoter. Putative promoter elements located within the US11 promoter (Fig. 1) and nested deletions generated within the US11 promoter are indicated. (B) Activation of the US11 promoter by the IE proteins. The US11 promoter and the indicated deletion mutants were cotransfected into HFFs with a construct (pSVH) expressing the IE gene region under the control of the MIEP. Cells were harvested 48 h after transfection and assessed for CAT activity. Activity is expressed relative to the wild-type promoter at 100%. Except for the d40 deletion, the average (± standard deviation) of at least three experiments is represented. Results for the d40 deletion represent data from two experiments. ∗, P < 0.001 (student’s t test).

FIG. 3

Activation of the US11 promoter mutants by the IE proteins. The US11 promoter and promoter variants containing mutations in either the CREB site (Cm), the ATF site (Am), or both (CAm) were assessed by cotransfection into HFFs with the IE proteins. Cells were harvested 48 h after transfection and assessed for CAT activity. Activity is expressed relative to the wild-type promoter at 100%. The average (± standard deviation) of two experiments is represented. ∗, P < 0.003.

FIG. 4

Gel shift analysis of proteins binding to the US11 CREB and ATF sites. Gel shift assays were performed with probes containing the CREB or ATF site from the US11 promoter. Extracts were obtained from uninfected HFFs. Competition analysis was performed with either a 10-, 25-, or 50-fold excess of cold competitor DNA consisting of either the wild-type binding site or the mutated version of this site. The image was generated with a Hewlett-Packard ScanJet IIcx with Hewlett-Packard HP Deskscan II software (version 2.3.1) and labeled with Microsoft PowerPoint.

FIG. 5

(A) Activation of US11 promoter deletions by pp71. pUS11dHCAT and the indicated deletion mutants were cotransfected into HFFs with pSVH and either pCMV71 or pCMV71dlPvuI as described for Fig. 2. Cells were harvested 48 h after transfection and assessed for CAT activity. The results are expressed as fold activation relative to the activation by the IE proteins in the presence of the control plasmid pCMV71dlPvuI and represent the average of three independent experiments. ∗, P = 0.01. (B) Activation of the US11 promoter mutants by the pp71 tegument protein. The US11 promoter and promoter variants containing mutations in either the CREB site (Cm), the ATF site (Am), or both (CAm) were assessed by cotransfection into HFFs with the IE proteins and either pCMV71 or pCMV71dlPvuI as described for Fig. 3. Cells were harvested 48 h after transfection and assessed for CAT activity. The results are expressed as fold activation relative to the activation by the IE proteins in the presence of the control plasmid pCMV71dlPvuI and represent the average of three independent experiments. ∗, P ≤ 0.05.

FIG. 6

Northern blot analysis of recombinant HCMV. HFFs were infected with 5 PFU of the indicated viruses per cell, harvested at the indicated times, and assessed for US11 RNA levels by Northern blot analysis utilizing a ³²P-labeled probe to the US11 gene. RNA levels were quantitated by PhosphorImager analysis. (A) Representative Northern blot analysis of the 1.5-kb US11 mRNA in cells infected with the recombinant viruses, as well as US11 mRNA levels in AD169-infected cells. Values shown in panel B (average of results from two replicate experiments) were corrected for multiplicities of infection by stripping the blot and reprobing with a probe to the UL99 gene and expressed relative to the level of US11 mRNA expression in AD169-infected cells at 8 h after infection.