Functional properties of the human cytomegalovirus IE86 protein required for transcriptional regulation and virus replication

Abstract

The human cytomegalovirus (HCMV) IE86 protein is essential for HCMV replication due to its ability to transactivate critical viral early promoters. In the current study, we performed a comprehensive mutational analysis between amino acids (aa) 535 and 545 of IE86 and assessed the impact of these mutations on IE86-mediated transcriptional activation. Using transient assays and complementing analysis with recombinant HCMV clones, we show that single amino acid mutations differentially impair the ability of IE86 to mediate transactivation of essential early gene promoters. The conserved tyrosine at amino acid 544 is critical for activation of the UL54 promoter in vitro and in the context of the viral genome. In contrast, mutation of the proline at position 535 disrupted activation of the UL54 promoter in transient assays but displayed activity similar to that of wild-type (WT) IE86 when assessed in the genomic context. To examine the underlying mechanism of this differential effect, glutathione S-transferase (GST) pulldown assays were performed, revealing that Y544 is critical for binding to the TATA binding protein (TBP), suggesting that this interaction is likely necessary for the ability of IE86 to activate the UL54 promoter. In contrast, mutation of either P535 or Y544 disrupted activation of the UL112-113 promoter both in vitro and in vivo, suggesting that interaction with TBP is not sufficient for IE86-mediated activation of this early promoter. Together, these studies demonstrate that IE86 activates early promoters by distinct mechanisms.

FIG. 1.

Effect of IE86 mutations on UL54, UL112-113, and MIE promoter regulation. The pUL54-Luc (A), pUL112-113-Luc (B), or pMIEP-Luc (C) reporter construct was cotransfected into primary fibroblasts with the pSVHk vector that expresses either wild-type IE86 or the indicated mutant in the context of the MIE gene region. Cell extracts were assessed for luciferase activity 48 h after transfection. Data are the average and standard deviation (SD) of results of a minimum of two experiments performed in duplicate, normalized to promoter activity in the presence of wild-type IE86. (D) Total protein was harvested at 48 h posttransfection and separated on a 10% SDS-PAGE gel. Western blot analysis was performed using a peptide antibody that recognizes the IE86 protein. An antibody to actin was included to normalize for protein loading. Symbols: *, significantly different from wild type, P < 0.05; C, level of promoter activity in the presence of the pSV0d empty vector control plasmid.

FIG. 2.

Effect of IE86 mutations on promoter activation, IE2 mRNA, and IE86 protein expression. (A) The pMIEP-Luc, pUL54-Luc, or UL112-113-Luc reporter construct was cotransfected into primary fibroblasts with pMCRS86 vector expressing either wild-type IE86 or the indicated mutant. Cell extracts were assessed for luciferase activity 48 h after transfection. Data are the average and SD of results of a minimum of two experiments performed in duplicate, normalized to promoter activity in the presence of wild-type IE86, and presented on a log scale to assist in the comparison of promoter activation levels. Symbols: *, significantly different from wild type, P < 0.05; C, level of promoter activity in the in the presence of the pSV0d empty vector control plasmid. (B and C) Primary fibroblasts were transfected with the pSVHk vector expressing either wild-type IE86 or the indicated mutant. (B) Total RNA was harvested at 48 h posttransfection, and real-time RT-PCR analysis performed to determine IE2 mRNA levels. Analyses were performed in duplicate, and relative expression normalized to GAPDH mRNA levels was expressed as mean ± SD. (C) Total protein was harvested at 48 h posttransfection and separated on a 12.5% SDS-PAGE gel. Western blot analysis was performed using an antibody that recognizes IE72 and IE86. An antibody to actin was included to normalize for protein loading. Symbol: *, nonspecific band.

FIG. 3.

Analysis of mutant HCMV BACs. (A) Schematic of the MIE gene region of the mutant BACs. The Zeocin resistance cassette containing a SmaI site is represented by the gray box. The location of primer sequences used to confirm appropriate recombination are also indicated by arrows. (B) Restriction enzyme analysis of HCMV BACs was performed using SalI and HindIII. Following digestion, the fragmented DNA was run on a 0.8% agarose gel. (C) PCR analysis of HCMV BACs was performed using the indicated primers, and the gel fragments were separated on a 1.2% agarose gel. The HCMV WT BAC results in an approximately 0.6-kb fragment, with the Zeocin resistance cassette increasing the size of this fragment to 1.1 kb. (D) Southern blot analysis of HCMV BACs digested with SmaI showing the presence of the additional SmaI site in the WT-Rev, P535A, and Y544A BAC constructs due to the presence of the Zeocin resistance cassette.

FIG. 4.

Replication kinetics of HCMV WT and WT-Rev virus. WT-Rev and mutant HCMV BACs were transfected into primary fibroblasts by calcium phosphate transfections. (A) DNA replication was assessed by isolating genomic DNA at the indicated times following transfection and assessment of gB DNA levels by real time PCR. (B) Growth kinetics were assessed by isolating genomic DNA from the culture supernatant at the indicated times following transfection and assessment of gB DNA levels by real-time PCR.

FIG. 5.

Regulation of viral gene expression in BAC-transfected cells. Total RNA was isolated from BAC-transfected cells at the indicated times posttransfection and analyzed by real-time RT-PCR using primers specific for the UL54 (A), UL112-113 (B), and IE2 (C) transcript levels. Data are expressed as mean ± SD from results of two independent experiments performed in duplicate. Symbol: *, significantly different from wild type, P < 0.05. (D) Total protein was harvested at 48 h posttransfection and separated on a 10% SDS-PAGE gel. Western blot analysis was performed using a peptide antibody that recognizes the IE86 protein. An antibody to actin was included to normalize for protein loading.

FIG. 6.

GST pulldown analysis of IE86 mutant proteins. (A) SDS-PAGE analysis of wild type and the indicated IE86 mutant proteins generated by in vitro transcription/translation (IVTT) reactions. Positive-control (+) and negative-control (−) IVTT reactions were included. (B) Wild-type or mutant IE86 proteins were incubated with bacterially expressed GST or GST-TBP. GST complexes were then purified by glutathione Sepharose beads, the bound proteins separated by SDS-PAGE, and the ³⁵S-labeled IE86 proteins visualized on a Typhoon imager.