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. 2001 Jul;75(13):6062-9.
doi: 10.1128/JVI.75.13.6062-6069.2001.

Identification of a novel transcriptional repressor encoded by human cytomegalovirus

L A LaPierre  1 , B J Biegalke
Affiliations

Identification of a novel transcriptional repressor encoded by human cytomegalovirus

L A LaPierre et al. J Virol. 2001 Jul.

Abstract

The expression of human cytomegalovirus (HCMV) genes during viral replication is precisely regulated, with the interactions of both transcriptional activators and repressors determining the level of gene expression. One gene of HCMV, the US3 gene, is transcriptionally repressed early in infection. Repression of US3 expression requires viral infection and protein synthesis and is mediated through a DNA sequence, the transcriptional repressive element. In this report, we identify the protein that represses US3 transcription as the product of the HCMV UL34 open reading frame. The protein encoded by UL34 (pUL34) binds to the US3 transcriptional repressive element in yeast and in vitro. pUL34 localizes to the nucleus and alone is sufficient for repression of US3 expression. The data presented here, along with earlier data (B. J. Biegalke, J. Virol. 72:5457-5463, 1998), suggests that pUL34 binding of the transcriptional repressive element prevents transcription initiation complex formation.

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Figures

FIG. 1
FIG. 1
Electrophoretic mobility shift assays. (A) Double-stranded radiolabeled DNA fragments containing either a tre (w) or a mutant version of the tre (m) were used to assay for specific DNA-binding proteins in nuclear extracts prepared from mock-infected (mock) or HCMV-infected (infected) HDFs. Lanes 1 to 4, US3 sequences from −25 to +10; lanes 5 to 6, sequences from −22 to +1; lanes 7 and 8, sequences from −58 to +32. (B) Specificity of DNA-protein interactions. Competition experiments were performed, using nuclear extracts from infected HDFs and adding either no competitor (lane 9) or a 100-fold molar excess of unlabeled DNA fragments containing the US3 tre (wt tre, lane 10), the mIE crs (crs, lane 11), or the mutant version of the tre (m tre, lane 12) to the DNA-protein binding reactions prior to addition of the radiolabeled tre-containing fragment. Arrows, specific DNA-protein interactions; ∗, unbound probe; dash, tre-independent DNA-protein interaction. (C) Sequence of the US3 regulatory region; the tre is underlined, the TATA box is indicated by a rectangle, nucleotide substitutions that create a nonfunctional tre (5) are indicated by asterisks. The locations of the −22 to +1, −25 to +10, and −58 to +32 probes are indicated; the bent arrow indicates the transcription start site.
FIG. 2
FIG. 2
(A) SDS-polyacrylamide gel electrophoresis of the in vitro-synthesized pUL34 and luciferase proteins. Lane 1, 5 μl of the in vitro translation reaction utilizing the UL34-encoding plasmid; lane 2, 5 μl of the translation reaction utilizing the luciferase-encoding (luc) plasmid; lane 3, control reaction containing no template plasmid. The positions of the molecular weight markers are indicated. (B) EMSA analysis of in vitro-synthesized pUL34. The radiolabeled double-stranded DNA probe used in lanes 1 to 8 consisted of a tre-containing fragment (−22 to +1, see Fig. 1C), while the probe in lanes 9 to 10 contained a mutant version of the tre. The proteins incubated with the DNA probes are as follows: lane 1, infected cell extracts (Inf.); lane 2, extracts from mock-infected cells supplemented with 1 μl of the in vitro-translated pUL34 (M+UL34); lane 3, 1 μl of the in vitro-translated pUL34 preincubated with a 200× molar excess of unlabeled tre-containing US3 sequences from +1 to −22 (competitor, c); lane 4, no protein; lane 5, 1 μl of the in vitro-translated luciferase protein (luc); lane 6, 1 μl of the in vitro-translated pUL34; lane 7, 5 μl of the in vitro-translated pUL34; lane 8, extracts from mock-infected cells (Mock); lane 9, no protein; and lane 10, 1 μl of the in vitro-translated pUL34. Arrow, specific DNA-protein interaction.
FIG. 3
FIG. 3
Micrographs of HDFs transfected with pEGFP-C2 (A and B) or pBJ507 (which expresses an EGFP-UL34 fusion protein) (C, D, E, and F). Cells were stained with DAPI and visualized with UV and FITC filters (×400 magnification). Panels A, C, and E, DAPI staining; panels B, D, and F, GFP fluorescence. Arrows indicate the positions of the transfected cells.
FIG. 4
FIG. 4
Analysis of the effect of pUL34 on US3 transcription. Reporter gene plasmids that express the lacZ gene under the control of the US3 enhancer and promoter and contain either the tre (pBJ171, open columns) or a mutant version of the tre (pBJ214, crosshatched columns) were transfected into HDFs alone or with plasmids that express IE1 (pEQ273), IE2 (pEQ326), or IE1 and IE2 proteins (pEQ276) or pUL34 (pBJ386) as indicated. Reporter gene activity was assayed ∼36 h after transfection, by measuring the fluorescence of the cleavage product of the β-galactosidase substrate, MUG. The amount of plasmid DNA was kept constant in the transfections by adding the appropriate amount of a control plasmid, pBJ201, which contains the mIE promoter but expresses no protein (4). Background levels of β-galactosidase obtained with a promoterless lacZ-containing plasmid, pEQ3, were subtracted from the values obtained with pBJ171 and pBJ214. (A) pUL34 repression of US3 expression. The data presented are the averages of duplicate transfections plus one standard deviation. (B) Comparison of the repressive effect of infection with the repressive effect of pUL34 on IE1 and IE2 activation of the US3 promoter and tre.
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References

    1. Ahn K, Angulo A, Ghazal P, Peterson P A, Yang Y, Fruh K. Human cytomegalovirus inhibits antigen presentation by a sequential multistep process. Proc Natl Acad Sci USA. 1996;93:10990–10995. - PMC - PubMed
    1. Anders D G, Kacica M A, Pari G S, Punturieri S M. Boundaries and structure of human cytomegalovirus oriLyt, a complex origin for lytic-phase DNA replication. J Virol. 1992;66:3373–3384. - PMC - PubMed
    1. Biegalke B J. Regulation of human cytomegalovirus US3 gene transcription by a cis-repressive sequence. J Virol. 1995;69:5362–5367. - PMC - PubMed
    1. Biegalke B J. IE2 protein is insufficient for transcriptional repression of the human cytomegalovirus US3 promoter. J Virol. 1997;71:8056–8060. - PMC - PubMed
    1. Biegalke B J. Characterization of the transcriptional repressive element of the human cytomegalovirus immediate-early US3 gene. J Virol. 1998;72:5457–5463. - PMC - PubMed

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