Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Dec 7;101(49):17234-9.
doi: 10.1073/pnas.0407933101. Epub 2004 Nov 30.

Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation

Michael Nevels  1 , Christina Paulus, Thomas Shenk
Affiliations

Human cytomegalovirus immediate-early 1 protein facilitates viral replication by antagonizing histone deacetylation

Michael Nevels et al. Proc Natl Acad Sci U S A. .

Abstract

The human cytomegalovirus 72-kDa immediate-early (IE)1 and 86-kDa IE2 proteins are expressed at the start of infection, and they are believed to exert much of their function through promiscuous transcriptional activation of viral and cellular gene expression. Here, we show that the impaired growth of an IE1-deficient mutant virus in human fibroblasts is efficiently rescued by histone deacetylase (HDAC) inhibitors of three distinct chemical classes. In the absence of IE1 expression, the viral major IE and UL44 early promoters exhibited decreased de novo acetylation of histone H4 during the early phase of infection, and the hypoacetylation correlated with reduced transcription and accumulation of the respective gene products. Consistent with these findings, IE1 interacts specifically with HDAC3 within infected cells. We also demonstrate an interaction between IE2 and HDAC3. We propose that the ability to modify chromatin is fundamental to transcriptional activation by IE1 and, likely, IE2 as well.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
HDAC inhibitors overcome the growth defect of an IE1-deficient mutant HCMV. (A) Comparison of viral protein accumulation in MRC-5 cells infected with WT (Towne) and mutant (CR208) viruses in the absence or continuous presence (beginning 24 h before infection) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID50 per cell, and expression of IE1 and IE2 (IE1/2), as well as the late viral protein pp28, was monitored at 24–96 h postinfection (hpi) by Western blot assay. (B) Multistep growth analysis of WT (Towne, circles) and mutant (CR208, triangles) viruses in the absence (filled symbols) or continuous presence (open symbols) of 300 nM TSA. MRC-5 cells were infected at a multiplicity of 0.1 TCID50 per cell, and virus yields were monitored for 144 h. Symbols identify mean values from two experiments. (C) Titers of WT (Towne) and mutant (CR208) viruses in the absence or continuous presence of the HDAC inhibitors HC toxin (HCT, 100 nM), sodium butyrate (NaB, 1 mM), or TSA (300 nM). MRC-5 cells were infected at a multiplicity of 0.1 TCID50 per cell, and yields were determined 120 h later. Bars represent mean values and SEs from three separate infections. (D) HDAC inhibitors induce histone hyperacetylation. MRC-5 cells were treated with 100 nM HCT, 300 nM TSA, or 1 mM NaB for 24 h, and Western blot analyses were performed by using Abs against acetylated histone H3 (Ac-H3) or the unmodified protein (H3).
Fig. 2.
Fig. 2.
Pretreatment of cells with HDAC inhibitors can support normal IE1-mutant virus growth. (A) Effect of pretreatment with HDAC inhibitors on the titers of WT Towne (black bars) and CR208 mutant (white bars) viruses after infection at a multiplicity of 0.01, 0.1, or 1 TCID50 per cell. MRC-5 cells were treated with 100 nM HC toxin (HCT), 2 mM sodium butyrate (NaB), or 500 nM TSA for 24 h before infection, and virus titers were determined at 72 h after infection. Bars represent mean values from three separate infections with SEs. (B) Immunofluorescence analysis showing expression of the ppUL44 early viral protein at 16 h after infection in MRC-5 cells infected at a multiplicity of ≈1 TCID50 per cell with WT (Towne) or mutant (CR208) virus. Cells were pretreated with 500 nM TSA or the respective solvent dimethyl sulfoxide (w/o) for 24 h before infection. Cells in at least 10 fields of view (≈40 cells per field) were counted. Bars represent mean values with SEs presented as the percentage of 4′,6-diamidino-2-phenylindole-stained nuclei that also stained positive for ppUL44.
Fig. 3.
Fig. 3.
The HCMV major IE and UL44 promoters are regulated by histone acetylation and IE1. (A) Quantitative real-time RT-PCR was performed with total RNA isolated from MRC-5 cells at 6–72 h postinfection (hpi) with WT (Towne) or mutant (CR208) virus at a multiplicity of 0.1 TCID50 per cell by using UL44- or UL122 (IE2)-specific primers. (B) Luciferase assays were performed in MRC-5 cells, which were transfected with reporter plasmids pGL3-ICP36 (UL44) or pGL3-MIEP (major IE) and treated with 100 nM HC toxin at 24–40 h after transfection, as indicated. At 24 h after transfection, cells were infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID50 per cell for 16 h. (C) Luciferase assays were performed in which MRC-5 cells were cotransfected with reporter plasmids pGL3-ICP36 (UL44 promoter) or pGL3-MIEP (major IE promoter) and empty vector (w/o) or expression plasmids encoding HDAC3, IE1, or both. As indicated, cells were treated with 100 nM HC toxin (HCT), 2 mM sodium butyrate (NaB), or 500 nM TSA at 24–40 h after transfection. Transfections were performed in triplicate, and mean relative light units with SEs (× 103 for the UL44 promoter and × 106 for the major IE promoter) are presented.
Fig. 4.
Fig. 4.
Decreased histone H4 acetylation at the HCMV major IE and UL44 promoters in the absence of IE1. Chromatin immunoprecipitation assays were performed on chromatin from MRC-5 cells infected with WT (Towne) or mutant (CR208) viruses at a multiplicity of 1 TCID50 per cell by using an Ab specific to amino-terminally acetylated histone H4- and UL44-specific, major IE-specific, or c-fos-specific primers for quantitative real-time PCR of coprecipitated DNA. PCRs were performed in triplicate, and mean values normalized to input DNA levels are presented, representing changes in acetylation at 6 and 12 h postinfection (hpi) relative to 1 h postinfection (set at 1).
Fig. 5.
Fig. 5.
Physical interaction of IE1 and IE2 with HDAC3 in vivo.(A) Coimmunoprecipitation of IE1 or IE2 with epitope-tagged HDAC3 from transfected cells. H1299 cells were transfected with pcDNA-IE1, pcDNA-IE2, pcDNA-HDAC3-Flag, or empty vector as indicated, and immunoprecipitations were performed by using an anti-Flag agarose conjugate or a nonspecific agarose-Ab conjugate. Proteins from immunoprecipitates or whole-cell lysates were detected by Western blotting. (B) Coimmunoprecipitation of epitope-tagged HDAC3 with tagged IE1 or IE2 from transfected cells. H1299 cells were transfected with pCGN-IE1, pCGN-IE2, pcDNA-HDAC3-Flag, or empty vector as indicated, and immunoprecipitations were performed by using an anti-HA agarose conjugate or Sepharose beads without Ab, followed by Western blotting. IgG, Ig heavy chains. (C) Coimmunoprecipitation of IE1 or IE2 with endogenous HDAC3 from infected cells. MRC-5 cells were infected with HCMV (Towne) at a multiplicity of 5 TCID50 per cell, as indicated, and immunoprecipitations were performed by using an anti-HDAC3 mAb or empty beads. Proteins from immunoprecipitates or whole-cell lysates were detected by Western blotting. (D) Luciferase assays from cells cotransfected with pGL3-ICP36 (UL44) or pGL3-MIEP (major IE) and empty vector, pcDNA-IE2, pcDNA-HDAC3-Flag, or combinations thereof, as indicated. Transfections were performed in triplicate, and bars represent mean relative light units with SEs (× 103 for the UL44 promoter and × 106 for the major IE promoter).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Pass, R. F. (2001) in Fields Virology, eds. Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman, B. & Strauss, S. E. (Lippincott, Philadelphia), Vol. 2, pp. 2675–2705.
    1. Mocarski, E. S. & Courcelle, C. T. (2001) in Fields Virology, eds. Knipe, D. M., Howley, P. M., Griffin, D. E., Lamb, R. A., Martin, M. A., Roizman, B. & Strauss, S. E. (Lippincott, Philadelphia), Vol. 2, pp. 2629–2673.
    1. Castillo, J. P. & Kowalik, T. F. (2002) Gene 290, 19–34. - PubMed
    1. Marchini, A., Liu, H. & Zhu, H. (2001) J. Virol. 75, 1870–1878. - PMC - PubMed
    1. Heider, J. A., Bresnahan, W. A. & Shenk, T. E. (2002) Proc. Natl. Acad. Sci. USA 99, 3141–3146. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources