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. 2006 Mar 7;103(10):3840-5.
doi: 10.1073/pnas.0600007103. Epub 2006 Feb 23.

A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling

Christina Paulus  1 , Steffen Krauss, Michael Nevels
Affiliations

A human cytomegalovirus antagonist of type I IFN-dependent signal transducer and activator of transcription signaling

Christina Paulus et al. Proc Natl Acad Sci U S A. .

Abstract

Type I IFNs are crucial components of the innate immune response to viral attack. They are rapidly synthesized and secreted after infection with human cytomegalovirus (CMV) and trigger a signal transduction pathway that involves successive activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1) and STAT2. The activated STATs, together with the IFN regulatory factor 9 protein, form a trimeric transcription complex (IFN-stimulated gene factor 3) that stimulates expression of numerous IFN-responsive genes, many of which exhibit antiviral activity. Here we demonstrate that the viral 72-kDa IE1 protein (IE1-72kDa) confers partial resistance to the antiviral activity of type I IFNs upon CMV. Accordingly, IFN-responsive transcripts accumulate to substantially increased levels after infection with an IE1-deficient mutant as compared with wild-type virus, and ectopic expression of the viral protein in stably transfected cells is sufficient to block their induction. We further show that IE1-72kDa forms a physical complex with STAT1 and STAT2 in nuclei of infected cells and in vitro and prevents association of STAT1, STAT2, and IFN regulatory factor 9 with promoters of IFN-responsive genes in vivo. Our results indicate that the viral protein blocks an intranuclear step after nuclear translocation and before DNA binding of IFN-stimulated gene factor 3, presumably by interfering with the integrity and/or correct subnuclear localization of the protein complex. This study identifies the CMV IE1-72kDa protein as a viral antagonist of the cellular innate immune response, inhibiting IFN-dependent STAT signaling by means of an unprecedented molecular mechanism.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Differential effects of exogenous IFN-α or neutralization of endogenous IFN-β on wild-type (Towne) and IE1-deficient (CR208) mutant virus yields. Experiments were performed in triplicate, and bars represent mean values with standard errors. (A) MRC-5 cells treated with 250 or 1,000 units/ml recombinant IFN-α (+) or untreated cells (−) were infected at a multiplicity of 5 plaque-forming units (PFU) per cell. Virus yields were determined 72 h after infection. (B) MRC-5 cells treated with 500 neutralizing units/ml of an IFN-β-specific antiserum (+) or untreated cells (−) were infected at a multiplicity of 0.1 PFU per cell. Virus yields were determined 7 days after infection.
Fig. 2.
Fig. 2.
IE1 expression antagonizes induction of ISGs. (A) MRC-5 cells treated with 1,000 units/ml recombinant IFN-α (+) or untreated cells (−) were infected with wild-type (Towne) or mutant (CR208) CMV at a multiplicity of 1 PFU per cell or mock-infected. ISG54/MxA (Left) and IFN-β (Right) mRNAs were quantified 24 h or 6 h after infection, respectively, by kinetic RT-PCR. (B) MRC-5 cells were infected with wild-type (Towne) or mutant (CR208) CMV at a multiplicity of 1 PFU per cell, and ISG54 mRNA was quantified by kinetic RT-PCR at 6, 24, and 72 h after infection. (C) The cell lines ihfie1 (+IE1) and ihf (−IE1) were treated with 1,000 units/ml recombinant IFN-α (+) or were left untreated (−), and ISG54 or MxA mRNAs were quantified by kinetic RT-PCR.
Fig. 3.
Fig. 3.
IE1-72kDa fails to affect accumulation, phosphorylation, and subcellular localization of ISGF3 components. (A) Western blots showing steady-state levels of the indicated proteins in ihf or ihfie1 cells at various times before and after treatment with 1,000 units/ml recombinant IFN-α. (B) Indirect immunofluorescence images showing typical subcellular localization of the indicated proteins in ihf or ihfie1 cells before and after treatment for 1 h with 1,000 units/ml recombinant IFN-α. A rabbit-specific Alexa Fluor 488 conjugate (Molecular Probes) was used as secondary antibody. Before analysis, IE1 expression was determined by immunofluorescence to be present in ≈99% of ihfie1 cells. (Magnification: ×500.)
Fig. 4.
Fig. 4.
IE1-72kDa interferes with DNA association of ISGF3 components. (A) ChIP assays using STAT2-specific antibodies, quantified by real-time PCR. Amounts of DNA coprecipitated from ihf or ihfie1 cells at 0, 15, 30, or 60 min after IFN-α treatment (1,000 units/ml) were normalized to input DNA and plotted as mean values relative to nontreated samples (0 min, set to 1). (B) ChIP assays using STAT1-, STAT2-, or IRF9-specific antibodies. Amounts of DNA coprecipitated with polyclonal antibodies directed against the indicated proteins from ihf or ihfie1 cells at 60 min after IFN-α treatment (1,000 units/ml) were quantified by real-time PCR, normalized to input DNA, and plotted as mean values relative to nontreated samples (set to 1). (C) STAT2-specific ChIP assays from MRC-5 cells that were mock-treated or infected with wild-type Towne or CR208 viruses at a multiplicity of 1 PFU per cell for 12 h. Amounts of coprecipitated DNA were quantified by real-time PCR, normalized to input, and plotted as mean values relative to mock-infected samples (set to 1).
Fig. 5.
Fig. 5.
Nuclear colocalization of IE1-72kDa with ISGF3 proteins. Subconfluent ihfie1 cells were treated with 1,000 units/ml IFN-α for 1 h and subjected to double-labeling indirect immunofluorescence analysis using an IE1-specific mouse antibody in combination with rabbit sera directed against STAT2, STAT1, or IRF9, as indicated, followed by incubation with anti-mouse Alexa Fluor 546 and anti-rabbit Alexa Fluor 488 conjugates. Images were acquired on a Leica DMXR microscope. Typical nuclei showing dot-like, mitotic chromatin-associated or nuclear diffuse localization of IE1-72kDa and corresponding STAT2 (A), STAT1 (B), or IRF9 (C) stainings are shown. (Magnification: ×500.)
Fig. 6.
Fig. 6.
Physical interaction of IE1-72kDa with ISGF3 components. (A) In vivo binding analyzed by coimmunoprecipitation assay. MRC-5 cells were mock-infected or infected at a multiplicity of 3 PFU per cell with CMV Towne or CR208 for 24 h. Immunoprecipitations were performed by using an IE1-specific antibody or empty Sepharose beads. Proteins from whole-cell lysates (≈5% of material used as input for immunoprecipitations; Left) or immunoprecipitates (Right) were separated in SDS/10% polyacrylamide gels and detected by Western blotting with antibodies as indicated. IgG, Ig heavy chains. (B) In vitro binding analyzed by GST capture assay. Equal amounts of GST or GST-IE1 proteins were reacted with whole-cell extracts prepared from MRC-5 cells (+) or GST pull-down lysis buffer (−), and complexes or whole-cell lysates (Input) were separated in SDS/10% polyacrylamide gels. STAT1, STAT2, or IRF9 proteins were detected by Western blotting, and two different film exposures are shown.
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