Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression - PubMed (original) (raw)
Human cytomegalovirus infection causes degradation of Sp100 proteins that suppress viral gene expression
Young-Eui Kim et al. J Virol. 2011 Nov.
Abstract
The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.
Figures
Fig. 1.
Expression patterns of Sp100 proteins in HCMV-infected fibroblasts. (A) HFs were mock infected (M) or infected with intact or UV-inactivated Towne virus at the indicated MOIs. Cell lysates were prepared at 24 h postinfection and immunoblotted with Abs for Sp100, IE1/IE2, and β-actin. (B) HFs were mock infected or infected with Towne virus at an MOI of 1 or 10. Total RNAs were prepared at 24 h postinfection, and the levels of Sp100A transcripts were measured by quantitative real-time RT-PCR. The results shown are the mean values and standard errors of three independent experiments. (C) HFs were mock infected or infected at an MOI of 0.2, 1, or 5. Cells were thoroughly washed after virus adsorption to remove any unbound virus. The culture supernatants were obtained at 24 h and used to administer fresh cells. Total cell lysates were prepared from initially infected cells (left 4 lanes) and from cells treated with the supernatants for 24 h (right 4 lanes) and subjected to immunoblot analysis with anti-Sp100 or anti-IE1/IE2 Abs. (D) HFs were mock infected or infected with Towne at an MOI of 10. Cell lysates were prepared at the indicated time points, and immunoblotting was performed with Abs for Sp100, IE1/IE2, p52 (UL44), pp28 (UL99), and β-actin. Lanes M, mock infection.
Fig. 2.
Effect of CR208 virus infection on Sp100 protein levels. (A) HFs were infected with wild-type (Wt) Towne or mutant CR208 virus at an MOI of 10. Cell lysates were prepared at the indicated time points and subjected to immunoblotting using Abs for Sp100, IE1/IE2, p52, pp28, and β-actin. (B) Control HFs (shC) and cells expressing shRNA for PML (shPML) were mock infected or infected with CR208 at an MOI of 5. Total cell lysates were prepared at the indicated time points, and immunoblot analysis was carried out with Abs for Sp100, IE1/IE2, and β-actin. Lanes M, mock infection.
Fig. 3.
Effects of proteasome inhibitors on Sp100 protein loss during HCMV infection and changes of proteasome activity in virus-infected cells. (A) HFs were mock infected or infected with Towne virus at an MOI of 2 and harvested at 24, 48, or 72 h or treated with dimethyl sulfoxide as a control, MG132 (10 μM), lactacystin (0.5 μM), or epoxomicin (0.5 μM) at 72 h and harvested at 96 h. Cell lysates were prepared and immunoblot assays were carried out using Abs for Sp100, IE1/IE2, p52 (UL44), pp28 (UL99), and β-actin. (B) Proteasome activity in cells infected with wild-type, UV-inactivated, and CR208 viruses. HFs were mock infected or infected with wild-type, UV-inactivated, or CR208 viruses at an MOI of 2 for 72 h and either left untreated (closed bars) or treated (open bars) with 30 μM MG132 for 2 h before measuring proteasome activities in cell lysates. (Top) Proteasome activities are indicated as mean values, and error bars represent the SDs of three independent experiments. RFU, relative fluorescence units; (bottom) cell lysates were analyzed by immunoblotting with Sp100, IE1/IE2, and β-actin Abs. Note that the SUMO-modified form of Sp100A was barely detectable under the cell lysis conditions employed, due to rapid deSUMOylation by SUMO proteases. Lane M, mock infection.
Fig. 4.
Effect of Sp100 depletion on HCMV DNA replication and gene expression. (A) Total cell lysates were prepared from control HFs (shC) and cells expressing shRNA for Sp100 proteins (shSp100), and immunoblot analysis was performed with Abs for Sp100. The levels of β-actin, used as a loading control, are shown. (B) shC and shSp100 cells were mock infected or infected with Towne virus at an MOI of 1. Samples were obtained from the culture medium at 48 and 72 h postinfection, diluted, and assayed for viral titer (infectious units) using the infectious center assay (20). (C) shC and shSp100 cells were infected with Towne virus at an MOI of 3. At 24 h, cells were fixed and stained with anti-UL44 Abs. The percentages of infected cells showing only the nuclear diffuse pattern of UL44 are indicated as gray bars, whereas the percentages of cells exhibiting the nuclear focus or replication compartment patterns of UL44 on the nuclear diffuse background are indicated as black bars. (D) shC and shSp100 cells were mock infected or infected with recombinant HCMVs harboring a reporter gene (Pol-luciferase or pp28-luciferase) at an MOI of 1. Cell lysates were prepared at the indicated time points, and luciferase assays were carried out. The graphs indicate mean values, and the error bars represent the SDs of three independent experiments. Closed bars, assays in shC cells; open bars, assays in shSp100 cells. (E) Cell lysates (shC and shSp100) prepared from pp28-luciferase virus-infected cells (see panel D) were subjected to immunoblot analysis to quantify viral proteins such as IE1, IE2, p52, and pp28 with specific Abs. (F) shC and shSp100 cells were infected with CR208 virus at MOIs of 0.1 and 0.5. Samples were taken from the culture medium at 5 days postinfection, diluted, and assayed for virus titers (infectious units [IFU]).
Fig. 5.
Effects of Sp100 depletion on the levels of acetylated histones associated with the MIE and pp28 promoters. shC and ShSp100 cells were infected with Towne virus at an MOI of 2, and ChIP assays were carried out at 24 h using acetylated histone H4-specific Ab (αAc-H4) and RNA polymerase II Ab (αPol II). The amounts of coprecipitated DNA were quantified by real-time PCR and normalized to the input amount, and their changes are shown as graphs.
Fig. 6.
Interaction of IE1 with Sp100A. (A) Interaction of IE1 with Sp100 in yeast cells. The X-Gal filter assays were performed using yeast cells expressing both GAL4-DB/Sp100A fusion protein and GAL4-A/SUMO-1, GAL4-A/IE1, or GAL4/Sp100A fusion protein. The cells expressing GAL4-DB/Sp100A and GAL4-A were used as a negative control. The top three panels show positive interactions. The relative strength of each interaction was also shown by directly measuring β-galactosidase activity in yeast cell lysates. (B) HFs were mock infected or infected with Towne virus at an MOI of 2. Total cell lysates were prepared at 24 h postinfection and immunoprecipitated with anti-IE1 Ab (ch443) or control IgG as indicated, followed by immunoblotting (IB) with anti-Sp100 and anti-IE1 Abs. Total cell lysates were also immunoblotted with anti-IE1 and anti-Sp100 Abs to show the protein expression levels. (C) 293T cells were cotransfected with plasmids encoding Sp100A-HA (wild type or mutants) and Myc-IE1, as indicated. At 48 h, total cell lysates were prepared and immunoprecipitated with anti-Myc Ab, followed by immunoblotting with anti-HA Ab. Total cell lysates were also immunoblotted with anti-Myc or anti-HA Abs. (D) Structures of wild-type and mutant Sp100A proteins used. The positions for the regions responsible for dimerization, PML NB binding, HP1α binding, and the nuclear localization signal (NLS) are designated. All Sp100A constructs except for Δ408-480 harbor an HA tag at the C terminus, and Δ408-480 harbors both an NLS tag (open circle) and an HA tag at its C terminus. (E) In vitro GST pulldown assays. Bacterially purified GST or GST-IE1 immobilized to glutathione-Sepharose beads was incubated with _in vitro_-translated wide-type or Δ3-152 mutant Sp100A proteins (with the HA tag), and the bound proteins were fractionated on SDS–8% polyacrylamide gels and visualized by immunoblotting with anti-HA Ab. One-tenth of the Sp100A proteins used in each binding reaction were loaded as input controls. One-tenth of the GST or GST-IE1 proteins used in each reaction were visualized with Coomassie blue staining.
References
- Ahn J. H., Brignole E. J., III, Hayward G. S. 1998. Disruption of PML subnuclear domains by the acidic IE1 protein of human cytomegalovirus is mediated through interaction with PML and may modulate a RING finger-dependent cryptic transactivator function of PML. Mol. Cell. Biol. 18: 4899–4913 - PMC - PubMed
- Ahn J. H., Hayward G. S. 2000. Disruption of PML-associated nuclear bodies by IE1 correlates with efficient early stages of viral gene expression and DNA replication in human cytomegalovirus infection. Virology 274: 39–55 - PubMed
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