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. 2015 Mar 26;11(3):e1004785.
doi: 10.1371/journal.ppat.1004785. eCollection 2015 Mar.

Positive role of promyelocytic leukemia protein in type I interferon response and its regulation by human cytomegalovirus

Young-Eui Kim  1 , Jin-Hyun Ahn  1
Affiliations

Positive role of promyelocytic leukemia protein in type I interferon response and its regulation by human cytomegalovirus

Young-Eui Kim et al. PLoS Pathog. .

Abstract

Promyelocytic leukemia protein (PML), a major component of PML nuclear bodies (also known as nuclear domain 10), is involved in diverse cellular processes such as cell proliferation, apoptosis, gene regulation, and DNA damage response. PML also acts as a restriction factor that suppresses incoming viral genomes, therefore playing an important role in intrinsic defense. Here, we show that PML positively regulates type I interferon response by promoting transcription of interferon-stimulated genes (ISGs) and that this regulation by PML is counteracted by human cytomegalovirus (HCMV) IE1 protein. Small hairpin RNA-mediated PML knockdown in human fibroblasts reduced ISG induction by treatment of interferon-β or infection with UV-inactivated HCMV. PML was required for accumulation of activated STAT1 and STAT2, interacted with them and HDAC1 and HDAC2, and was associated with ISG promoters after HCMV infection. During HCMV infection, viral IE1 protein interacted with PML, STAT1, STAT2, and HDACs. Analysis of IE1 mutant viruses revealed that, in addition to the STAT2-binding domain, the PML-binding domain of IE1 was necessary for suppression of interferon-β-mediated ISG transcription, and that IE1 inhibited ISG transcription by sequestering interferon-stimulated gene factor 3 (ISGF3) in a manner requiring its binding of PML and STAT2, but not of HDACs. In conclusion, our results demonstrate that PML participates in type I interferon-induced ISG expression by regulating ISGF3, and that this regulation by PML is counteracted by HCMV IE1, highlighting a widely shared viral strategy targeting PML to evade intrinsic and innate defense mechanisms.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Effects of PML knockdown on ISG induction by IFNβ treatment or UV-HCMV infection.
(A-C) Control (shC) and PML-knockdown (shPML) HF cells (see S1A and S1B Figs) were mock-infected or infected with UV-HCMV at an MOI of 5 IFU (infectious units) per cell (A and B), or untreated or treated with IFNβ (1 x 103 units/ml) (C), for 8 h. Total RNAs were prepared and ISG54, CXCL10, and PKR mRNA were quantified by qRT-PCR. The amounts of mRNA in cells treated with INFβ or infected with UV-HCMV over those of untreated or mock-infected cells are presented as fold inductions. The results shown are the mean values and standard errors of at least three independent experiments. (D) HF cells were transfected with 20 nmole of control scrambled (siC) or PML-specific siRNA (siPML) twice at times 0 and 24 h. At 72 h, cells were mock-infected or infected with UV-HCMV and ISG54 mRNA levels were measured by qRT-PCR as described in (A). PML-knockdown was confirmed by immunoblotting (see S1C Fig). (E) shC and shPML HF cells transduced with empty retroviral vector or PML-I-expressing vector were mock-infected or infected with UV-HCMV and ISG54 mRNA levels were determined by qRT-PCR as described in (A). The amounts of mRNA in cells infected with UV-HCMV over those in mock-infected cells are presented as fold inductions. Refer to S1D Fig for the expression levels of PML-I in shPML cells. (F) shPML HF cells were cotransfected with 0.5 μg of the ISG54 ISRE-Luc reporter plasmid and 1 μg of empty vector or plasmid encoding myc-PML-I as indicated. At 24 h, cells were untreated or treated with IFNβ (1 x 103 units/ml) for 8 h, and luciferase reporter assays were performed. See S1E Fig for the expression levels of PML proteins.
Fig 2
Fig 2. Effects of PML knockdown on the levels of STAT1 and STAT2 and of their activated forms.
(A) shC and shPML HF cells were mock-infected (M) or infected with UV-HCMV at an MOI of 5 IFU per cell. Total cell lysates were prepared at the indicated time points and immunoblot analysis was performed with antibodies specific for IRF3 and its phosphorylated form. The level of β-actin was used as a loading control. (B and C) shC and shPML HF cells were uninfected or infected with UV-HCMV as in (A) (B), or shC and shPML 293 cells were untreated or treated with IFNβ (1 x 103 units/ml) (C). Total cell lysates were prepared at the indicated time points and immunoblot analysis was performed with antibodies specific for STAT1, STAT2, and their phosphorylated forms. (D) shPML HF cells transduced with empty retroviral vectors or retroviral vectors expressing PML-I were mock-infected or infected with UV-HCMV at an MOI of 3 for the indicated times. Total cell lysates were prepared and immunoblotted using antibodies specific for PML (PG-M3), STAT1, STAT2, and their activated forms. (E) shC and shPML HF cells were uninfected or infected with UV-HCMV and immunoblotted as in (B), except that the culture medium added after virus adsorption included anti-IFNAR antibody or control IgG (5 μg/ml) as indicated. (F) STAT1 and STAT2 mRNA levels in shC and shPML HF cells were measured by qRT-PCR. (G) shC and shPML HF cells were untreated or treated with cycloheximide (CXH) (200 μg/ml) for the indicated times. Total cell lysates were prepared and immunoblotted. (H) Plots of STAT2 levels in cycloheximide treated or untreated shC and shPML HF cells.
Fig 3
Fig 3. Association of PML with STAT1, STAT2, and HDAC1 on ISG54 and CXCL10 promoters after UV-HCMV infection.
(A) Normal HF cells were mock-infected (-) or infected with UV-HCMV (+) at an MOI of 3 IFU per cell for 8 h and co-IP assays were carried out. Total cell lysates were prepared and immunoprecipitated with anti-PML antibody (PG-M3) or mouse IgG as a negative control. Immunoprecipitated samples and whole cell lysates were subjected to SDS-PAGE and then immunoblotted with antibodies for STAT1, STAT2, HDAC1, HDAC2, and PML (PG-M3). (B) Co-IP assays were performed as described in (A) using cell lysates treated with nucleases. (C) HF cells were infected as described in (A) and ChIP assays were performed using anti-PML (PG-M3), anti-STAT2, anti-HDAC1, and anti-HDAC2 antibodies. PCR was performed to detect ISG54 and CXCL10 promoter DNAs. The sizes of the DNA fragments amplified from the ISG54 and CXCL10 promoter regions were 199 bp and 241 bp, respectively. A 100 bp DNA ladder was used as a size marker.
Fig 4
Fig 4. Effects of ectopic IE1 expression on the IFNβ-mediated ISG54 induction.
(A) HF cells transduced by control MIN retroviral vector (HF-MIN) or MIN-IE1 (HF-MIN-IE1) were untreated or treated with IFNβ (1 x 103 units/ml) for 8 h. Total RNAs were prepared and the mRNA levels of ISG54 and β-actin were determined by qRT-PCR. The IE1 protein levels in cells were determined by immunoblotting with anti-IE1 antibody (6E1). (B) HF cells were uninfected or infected with Ad-Trans alone or with Ad-Trans plus Ad-IE1 at a total MOI of 10 plaque forming units (PFU) per cell for 48 h. Cells were then incubated for 8 h in the absence or presence of IFNβ (1 x 103 units/ml). qRT-PCR and immunoblot assays were performed as described in (A). (C) Reporter assays using the ISG54 ISRE-luciferase (Luc) reporter construct. 293T cells were cotransfected with 0.5 μg of ISG54 ISRE-Luc reporter plasmid and 0.5 μg of plasmid encoding intact HA-IE1, HA-IE1(1–475), or HA-IE1(Δ421–475) or 1.5 μg of plasmid encoding HA-IE1(Δ290–320), HA-IE1(1–420), or HA-IE1(Δ290–320/Δ421–475) mutants as indicated. All samples for transfection were made up to the same total amount of DNA using empty vectors. At 24 h, cells were untreated or treated with 1 x 103 units/ml (left) or 1 x 104 units/ml of IFNβ in absence or presence of 1 μM tricostatin A (TSA) (right) for 8 h, and luciferase reporter assays were performed. The results shown are the mean values and standard errors of three independent experiments. The expression levels of IE1 proteins were determined by immunoblotting with anti-HA antibody. (D) HF cells stably expressing wild-type or mutant IE1 (produced using retroviral vectors) were untreated or treated with IFNβ (1 x 103 units/ml) for 8 h. Total RNAs were prepared and the amounts of ISG54 mRNA were determined by qRT-PCR. The results shown are the mean values and standard errors of three independent experiments. The amounts of mRNA in cells treated with IFNβ compared to that in untreated cells are shown as fold inductions. The expression levels of IE1 were determined by immunoblotting using anti-IE1/IE2 antibody (810R).
Fig 5
Fig 5. Growth properties of the mutant virus encoding IE1(Δ290–320).
(A) HF and HF-IE1 cells were transfected via electroporation with T-BAC DNAs encoding wild-type IE1, IE1(Δ290–320) mutant, or its revertant. GFP images were taken at 12 days after transfection. (B) HF cells were infected with wild-type, IE1(Δ290–320) mutant, or revertant virus at an MOI of 0.1 or 3 IFU per cell. The growth curve shown represents the total number of infectious center-forming units in culture supernatants at the indicated sampling times. (C) HF cells were infected with recombinant viruses [wild-type (Wt), IE1(Δ290–320) mutant, or revertant (R)] at an MOI of 3. Total cell lysates were prepared at the indicated times, and immunoblotting was performed using anti-IE1/IE2 (810R), anti-p52, or anti-β-actin antibodies. (D) The stability of IE1(Δ290–320) in virus-infected cells. HF cells were infected with wild-type or IE1(Δ290–320) virus at an MOI of 3. At 6 h post-infection, cells were untreated or treated with 200 μl per ml of cycloheximide (CHX) or with 200 μl per ml of CHX plus 0.5 μM of MG132, and then further incubated for the indicated times. Cells were harvested and immunoblot assays were carried out using anti-IE1 (6E1) or anti- β-actin antibodies. β-Actin was used as a loading control. (E-H) Comparison of the effects of wild-type or mutant IE1expression on type I IFN response during virus infection. HF cells were mock-infected or infected with wild-type, IE1(Δ290–320), IE1(Δ421–475), and CR208 viruses, which were grown in IE1-expressing cells, or UV-HCMV at an MOI of 3. At 12 h post-infection, total RNAs were prepared and ISG54, CXCL10, or IFNβ mRNA levels were determined by qRT-PCR. The results shown are the mean values and standard errors of three independent experiments (E). The comparable expression of IE1 in recombinant virus-infected cells at 12 h after infection was shown by performing IFA (F) and immunoblotting (G) using two different anti-IE1 antibodies, 810R and 6E1, respectively. The genome copies of input viruses in cells immediately after virus adsorption (at time zero) were determined by qPCR (H).
Fig 6
Fig 6. Association of IE1 with STAT1, STAT2, HDAC1, HDAC2, and PML during infection and the binding of IE1(Δ290–320) with STAT2 and HDACs.
(A) HF cells were mock-infected (-) or infected (+) with HCMV (Towne) at an MOI of 2 IFU per cell. At 24 h after infection, total cell lysates were prepared and immunoprecipitated with anti-IE1 antibody (CH443); immunoprecipitation with mouse IgG was used as a negative control. The samples were subjected to SDS-PAGE and immunoblotted with antibodies for the indicated proteins (left panels). Immunoblot assays were also performed with total cell lysates to confirm protein expression levels (right panels). (B and C) HF cells were mock-infected or infected with wild-type, IE1(Δ290–320), or IE1(Δ421–475) mutant virus at an MOI of 2. At 12 h, total cell lysates were prepared and immunoprecipitated with anti-IE1 antibody (CH443) and immunoblotted with anti-STAT2 antibody (B) or with anti-HDAC1 or anti-HDAC2 antibodies (C). Total cell lysates were also immunoblotted with antibodies for IE1 (CH443), STAT2, HDAC1, and HDAC2 to confirm protein expression levels. (D) 293T cells were cotransfected with plasmids expressing HA-tagged wild-type, Δ290–320, or Δ290–320/Δ421–475 IE1 and myc-HDAC1, myc-HDAC2, myc-HDAC3, or myc-HDAC6 as indicated. At 48 h, whole cell lysates were prepared and immunoprecipitated with anti-Myc antibody and immunoblotted with anti-HA antibody. Levels of HA-IE1 and myc-HDAC proteins in whole cell lysates were determined by immunoblotting.
Fig 7
Fig 7. Comparison of the levels of the total and activated forms of STAT1 and STAT2, and the association of PML, STAT2, and HDAC1 with ISG promoters during wild-type or IE1 mutant virus infection.
(A) HF cells were infected with wild-type, IE1(Δ290–320), or IE1(Δ421–475) virus, at an MOI of 3 IFU per cell for the indicated times. Total cell lysates were prepared immunoblotted with antibodies for STAT1, STAT2, their phosphorylated forms, and IE1 (6E1). (B) HF cells were mock-infected or infected with wild-type, IE1(Δ290–320), IE1(Δ421–475) virus or UV-HCMV at an MOI of 3 for 12 h. ChIP assays were performed with anti-PML (PG-M3), anti-STAT2, anti-HDAC1 antibodies or with control IgG to detect the amounts of these proteins bound to ISG54 and CXCL10 promoters. The sizes of DNA fragments amplified from ISG54 and CXCL10 promoters were 199 bp and 241 bp, respectively. The 100 bp DNA ladder size markers are shown. (C) Control (MIN) and IE1 (wild-type or mutant)-expressing HF cells were mock-infected or infected with UV-HCMV at an MOI of 3 for 24 h and ChIP assays were performed as described in (B). (D) Model for the presence of a positive feedback loop for PML expression, the roles of PML in intrinsic defense and type I IFN signaling, and their regulation by HCMV IE1.
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This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIP) (2012R1A2A2A01002551). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.