A cytomegaloviral protein reveals a dual role for STAT2 in IFN-{gamma} signaling and antiviral responses

Abstract

A mouse cytomegalovirus (MCMV) gene conferring interferon (IFN) resistance was identified. This gene, M27, encodes a 79-kD protein that selectively binds and down-regulates for signal transducer and activator of transcription (STAT)-2, but it has no effect on STAT1 activation and signaling. The absence of pM27 conferred MCMV susceptibility to type I IFNs (alpha/beta), but it had a much more dramatic effect on type II IFNs (gamma) in vitro and in vivo. A comparative analysis of M27(+) and M27(-) MCMV revealed that the antiviral efficiency of IFN-gamma was partially dependent on the synergistic action of type I IFNs that required STAT2. Moreover, STAT2 was directly activated by IFN-gamma. This effect required IFN receptor expression and was independent of type I IFNs. IFN-gamma induced increasing levels of tyrosine-phosphorylated STAT2 in M27(-) MCMV-infected cells that were essential for the antiviral potency of IFN-gamma. pM27 represents a new strategy for simultaneous evasions from types I and II IFNs, and it documents an unknown biological significance for STAT2 in antiviral IFN-gamma responses.

Figure 1.

Inhibition of IFN-α–dependent luciferase induction by MCMV M27. (A) 3T3-ISRE-luc cells were infected with 10 PFU/cell MCMV-WT strain Smith and stimulated with 500 U/ml IFN-α for 5 h at the indicated time points p.i. Results were expressed as the percentage of luciferase activity in comparison with uninfected cells. The influence of viral gene expression was studied by the analysis of luciferase activity in the presence of PAA, which inhibits the expression of viral late gene expression, and by the analysis of UV-inactivated virus (IAV). (B) 3T3-ISRE-luc cells were infected with MCMV-WT, ΔM27-MCMV, or M27rev (10 PFU/cell each) and stimulated with 500 U/ml IFN-α for 5 h at 8 h (mock) or 29 h (MCMV-WT and ΔM27-MCMV) p.i. Luciferase activity was determined luminometrically as relative light units (RLU). Each value represents the mean ± SD from three independent experiments. (C) Expression kinetics of the M27 protein. MEFs were infected with 10 PFU/cell M27HA, and cell lysates were prepared at the indicated time points. Equivalent amounts of lysate were subjected to SDS-PAGE and then immunoblotted using anti-HA antibodies. The selective synthesis of MCMV IE proteins was achieved by infecting cells in the presence of 50 μg/ml of cycloheximide, which was then replaced by 5 μg/ml of actinomycin for a further 2 h. 250 μg/ml PAA was used to inhibit late phase gene expression.

Figure 2.

Inhibition of ΔM27-MCMV replication by IFN-α and IFN-γ. (A) Primary C57BL/6-MEFs were infected with MCMV-WT, ΔM27-MCMV, and M27rev (0.01 PFU/cell each). Titers of infectious virus were determined by a standard plaque assay. Growth curves were obtained after 48 h of preincubation of cells without IFN, 500 U/ml IFN-α, or 500 U/ml IFN-γ. (B) C57BL/6-MEFs were treated with graded concentrations of IFN-γ and infected as in A with MCMV-WT and ΔM27-MCMV (0.01 PFU/cell each). Viral titers obtained at 96 h p.i. were shown in relation to doses of IFN-γ.

Figure 3.

pM27 selectively affects STAT2. (A) pM27 down-regulates STAT2. C57BL/6-MEFs were either mock infected or infected with MCMV-WT, ΔM27-MCMV, or M27HA (10 PFU/cell each) for 24 h. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for STAT2, STAT1, IRF9/p48, IRF1, MCMV IE1/pp89, and β-actin. (B) NIH 3T3 were either mock infected, infected with VV-WT as a control, or infected with rVVM27FL (5 PFU/cell each). Cell lysates were prepared 16 h p.i. and analyzed by Western blot for STAT2, STAT1, pM27, and β-actin. (C) pM27 forms a complex with STAT2. STAT2^−/− fibroblasts were infected with rVV expressing M27FL or STAT2-HA, or coinfected with both viruses (3 PFU/cell each). Cell lysates were prepared 8 h p.i. using an EMSA buffer, split into two aliquots, and subjected to immunoprecipitation using anti-HA and anti-FLAG antibodies, respectively. Immunoprecipitates were analyzed by Western blot for STAT2, pM27, and β-actin.

Figure 4.

pM27 does not affect STAT1 but requires STAT2 for the inhibition of IFN-γ responses. (A) C57BL/6 MEFs and STAT2-deficient fibroblasts were infected with MCMV-WT or ΔM27-MCMV (10 PFU/cell each) for 24 h and exposed to 200 U/ml IFN-γ for 20 min. Equal protein amounts from 1:1 mixtures of nuclear and cytoplasmic cell extracts were evaluated by EMSA with a GAS probe (reference 21). Supershifting was performed by the addition of a STAT1-specific antibody before incubation with the probe. The mobility of STAT1 homodimers is indicated. (B) STAT2^−/− fibroblasts were preincubated with 500 U/ml IFN-γ for 48 h, or not incubated before infection, with MCMV-WT and ΔM27-MCMV (0.01 PFU/cell each) for the indicated time. Titers of progeny virus were determined by a standard plaque assay.

Figure 5.

MCMV replication in IFN-β–deficient cells. (A) Primary IFN-β–deficient MEFs were infected with MCMV-WT and ΔM27-MCMV (0.01 PFU/cell each). Titers of the infectious virus were determined by a standard plaque assay. Growth curves were obtained after 48-h preincubation of cells without IFN, or with 500 U/ml IFN-α, 500 U/ml IFN-γ, or a combination of 100 U/ml IFN-α and 100 U/ml IFN-γ. (B) Primary IFN-β–deficient MEFs were treated with 500 U/ml IFN-γ in combination with graded concentrations of IFN-α and infected with MCMV-WT and ΔM27-MCMV (0.01 PFU/cell each). Viral titers obtained at 96 h p.i. were shown in relation to doses of IFN-α. The titers in the presence of IFN-γ and in the complete absence of type I IFN are highlighted by arrows.

Figure 6.

Phosphorylation of STAT2 in response to IFN-γ. (A) Primary IFNAR1-deficient MEFs were either mock infected or infected with MCMV-WT or ΔM27-MCMV (10 PFU/cell each) for 24 h before being exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of cell lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr⁶⁸⁹ STAT2, and reprobed for STAT2, p-Tyr⁷⁰¹ STAT1, STAT1, and β-actin. (B) Primary IFNAR1- and IFNGR-deficient MEFs were either left untreated or treated with IFN-α (100 U/ml and 10 U/ml, respectively) or 500 U/ml IFN-γ for 20 min. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr⁶⁸⁹ STAT2 and reprobed for STAT2 and β-actin. (C) NIH 3T3 fibroblasts and J774 macrophages were either left untreated or treated with 50 U/ml IFN-α or 500 U/ml IFN-γ for 20 min. 50 NU/ml type I neutralizing antibodies were added as indicated to remove endogenously produced IFN-β. Equivalent amounts of cell lysates were analyzed by Western blot for p-Tyr⁶⁸⁹ STAT2, and reprobed for STAT2, p-Tyr⁷⁰¹ STAT1, and β-actin. (D) NIH 3T3 fibroblasts and IFNAR-deficient MEFs were not exposed or exposed to 500 U/ml IFN-γ or 50 U/ml IFN-α for 20 min. Equal protein amounts from 1:1 mixtures of nuclear and cytoplasmic cell extracts were evaluated by EMSA with an ISRE probe (reference 47). Supershifting was performed by the addition of a STAT2-specific antibody before incubation with the probe. The mobility of ISGF3 is indicated in the right margin.

Figure 7.

IFN-γ–induced increase of STAT2-P in ΔM27-infected cells and its impact for the inhibition of MCMV replication. (A) IFNAR1-deficient MEFs were either mock infected or infected with ΔM27-MCMV or MCMV-WT (10 PFU/cell each) for 24 h before exposed to 500 U/ml IFN-γ for the indicated time. Equivalent amounts of nucleoplasmic lysates were subjected to SDS-PAGE and analyzed by Western blot for p-Tyr⁶⁸⁹ STAT2, and reprobed for STAT2 and β-actin. (B) Comparative analysis of MCMV replication in MEF lacking components of the IFN signaling cascade. The indicated cells were incubated with 500 U/ml IFN-α or 500 U/ml IFN-γ for 48 h or left untreated before being infected with MCMV-WT or ΔM27-MCMV (0.01 PFU/cell each). The efficiency of MCMV replication is expressed as the ratio of virus yield at 96 h p.i., as determined by a standard plaque assay.

Figure 8.

Analysis of ΔM27-MCMV and M27rev in vivo. (A) C57BL/6, (B) 129Sv(ev) IFNGR1^−/− mice (reference 25), and (C) IFNAR1^−/− mice backcrossed with C57BL/6 mice were infected i.p. with 2 × 10⁵ PFU of tissue culture–derived ΔM27-MCMV or M27rev. Mice were killed at days 3, 7, 14, and 21, and organ titers of ΔM27-MCMV or M27rev in the liver and SGs were determined by a standard plaque assay. Each value represents the median of total SG titers or 1 g of liver from five infected animals. The dotted line shows the detection limit of organ titration (<100 PFU/gram of organ). (D) IFNAR1^−/− and IFNGR1^−/− mice (n = 10 per group) were infected i.v. with 5 × 10⁶ PFU of the indicated virus and monitored for survival over time.