The nucleocytoplasmic rabies virus P protein counteracts interferon signaling by inhibiting both nuclear accumulation and DNA binding of STAT1

Abstract

Rabies virus P protein inhibits alpha interferon (IFN-alpha)- and IFN-gamma-stimulated Jak-STAT signaling by retaining phosphorylated STAT1 in the cytoplasm. Here, we show that P also blocks an intranuclear step that is the STAT1 binding to the DNA promoter of IFN-responsive genes. As P is a nucleocytoplasmic shuttling protein, we first investigated the effect of the cellular distribution of P on the localization of STAT1 and consequently on IFN signaling. We show that the localization of STAT1 is correlated with the localization of P: in cells expressing a nuclear form of P (the short P3 isoform or the complete P in the presence of the export inhibitor leptomycin B), STAT1 is nuclear, whereas in cells expressing a cytoplasmic form of P, STAT1 is cytoplasmic. However, the expression of nuclear forms of P inhibits the signaling of both IFN-gamma and IFN-alpha, demonstrating that the retention of STAT1 in the cytoplasm is not the only mechanism involved in the inhibition of IFN signaling. Electrophoretic mobility shift analysis indicates that P expression in the cell extracts of infected cells or in stable cell lines prevents IFN-induced DNA binding of STAT1. The loss of the DNA binding of STAT1 and ISGF3 was also observed when purified recombinant P or P3 was added to the extracts of IFN-gamma- or IFN-alpha-treated cells, indicating that P directly affects the DNA binding activity of STAT1. Then products of the rabies virus P gene are able to counteract IFN signaling by creating both cytoplasmic and nuclear blocks for STAT1.

FIG. 1.

Rabies virus P protein is a nucleocytoplasmic protein. (A) P contains a CRM1-dependent NES between the residues 48 and 59 (underlined) and a conformational NLS in the carboxy-terminal part of P containing a short lysine-rich stretch located in close proximity to arginine 260 (²¹¹KKYK²¹⁴-R²⁶⁰) (20). P3 (residues 53 to 297) is an original product translated from the P gene and is present in infected cells (7); the first methionine (bold) of P3 is located inside the NES. The protein PΔN44 contains nine more residues than does P3 and also contains the NES. (B) Interaction of P3 and PΔN44 with STAT1 by a two-hybrid system. L40 yeast cells expressing the indicated bait and prey pairs were streaked onto plates lacking tryptophan and leucine. The induction of the lacZ reporter gene was assayed by the appearance of blue colonies as previously described by Vidy et al. (28).

FIG. 2.

Localization of pSTAT1 is correlated with the localization of P. (A) U373-MG control (upper panel) or P-expressing cell lines were incubated in the absence (middle panel) or presence of LMB (+LMB) at a final concentration of 20 nM for 1.5 h (lower panel). Cells were unstimulated or stimulated with 2,000 U/ml of hIFN-α or IFN-γ for 30 min as indicated, and they were fixed, permeabilized, and then stained with anti-P and anti-pSTAT1 antibodies and DAPI. (B) U373-MG cells were transfected with plasmids expressing P-GFP, P3-GFP (PΔN52-GFP), or PΔN44-GFP. Forty-eight hours after transfection, cells were treated with 2,000 U/ml of hIFN-α or hIFN-γ for 30 min as indicated. Cells were then stained with anti-P and anti-pSTAT1 antibodies and DAPI.

FIG. 3.

Localization of total STAT1 is not sensitive to LMB and depends on the P localization. (A) U373-MG cells transfected with plasmids expressing P-GFP were incubated in the absence (upper panel) or presence of LMB (+LMB) (lower panel) at a final concentration of 20 nM for 1.5 h and then treated with 2,000 U/ml of hIFN-α or hIFN-γ for 30 min as indicated. Cells were then stained with anti-P and anti-STAT1 antibodies and DAPI. The scale bars correspond to 40 μM. (B) U373-MG cells were incubated in the absence or presence of LMB (+LMB) at a final concentration of 20 nM for 1.5 h and then untreated (−) (left panel) or treated (+) (right panel) with 2,000 U/ml of hIFN-α for 30 min. Cells were then stained with anti-STAT1 antibodies and DAPI.

FIG. 4.

Expression of the nuclear forms of P inhibits IFN-α and IFN-γ signaling. (A) U373-MG cells were transfected with an ISRE-firefly luciferase reporter plasmid (pISRE-f. luc) and a Renilla luciferase expression vector (pTK-r.luc) and either an empty vector or a plasmid expressing P-GFP, P3-GFP, or PΔΝ44-GFP as indicated. At 48 hours after transfection, cells were untreated (−) or treated (+) with 2,000 U/ml of hIFN-α for 6 h prior to lysis and luciferase assays. (B) U373-MG cell lines expressing P or a control plasmid were transfected with pISRE-f.luc and pTK-r.luc. At 48 hours after transfection, cells were incubated in the absence or presence of LMB at a final concentration of 20 nM for 1 h. Cells were then unstimulated (−) or stimulated (+) with 2,000 U/ml of hIFN-α for 6 h prior to lysis and luciferase assays. (C) Same as described for panel B, but an IFN-γ-responsive GAS luciferase reporter was used instead of ISRE luciferase (pGAS-luc) and hIFN-γ was used instead of hIFN-α. All bars represent average values of firefly luciferase from triplicate samples, normalized to the expression of Renilla luciferase and expressed as percentages of IFN-stimulated controls; error bars indicate standard deviations. (D) U373-MG control or P-expressing cell lines were untreated (−) or treated with 2,000 U/ml of hIFN-γ (+γ) or hIFN-α (+α) for 24 h. The expression of PML, PKR, and IRF1 was studied by Western blot analysis with specific antibodies.

FIG. 5.

Effect of P expressed in infected cells and in P-expressing cell lines on the formation of GAF complexes. (A) U373-MG cells were not infected (−) (lanes 1 and 2) or infected (+) (lanes 3 and 4). Twenty-four hours after infection, cells were not treated (−) (lanes 1 and 3) or treated (+) (lanes 2 and 4) with 2,000 U/ml of hIFN-γ for 30 min. Total cell lysates were analyzed by EMSA using a GAS γ-³²P-labeled probe and native PAGE (upper panel). The same cell extracts were analyzed by immunoblotting with anti-pSTAT1 and anti-tubulin antibodies (lower panel). In addition, extracts of noninfected and IFN-γ-treated cells were incubated with anti-STAT1 antibodies prior to incubation with GAS γ-³²P-labeled probe (lane 5). The GAF complex and the supershifted GAF complex-IgG are indicated. (B) Control and P-expressing cell lines were not treated (−) (lanes 1 and 3) or treated (+) (lanes 2 and 4) with 2,000 U/ml of hIFN-γ for 30 min. Total cell lysates were analyzed by EMSA using a GAS γ-³²P-labeled probe and native PAGE. The same cell extracts were analyzed by immunoblotting with anti-pSTAT1 and anti-tubulin antibodies (lower panel).

FIG. 6.

Effect of recombinant P and P3 proteins on the formation of GAF complexes. Total cell extracts of hIFN-γ-treated U373-MG cells were mixed with increasing concentrations of His-tagged P (A) or His-tagged P3 (B) protein or unrelated His-tagged Gp17 protein (C) and submitted to EMSA analysis. (D) The same cell extracts were analyzed by immunoblotting with anti-pSTAT1 and anti-tubulin antibodies.

FIG. 7.

Effect of recombinant P and P3 proteins on the formation of ISGF3 complexes to the ISRE promoter. (A) U373-MG cells were not treated (−) (lane 1) or treated (+) (lanes 2 to 7) with 2,000 U/ml of hIFN-α for 30 min. Nuclear cell extracts were mixed with 1 μM of His-tagged P (lane 5), His-tagged P3 (lane 6), or His-tagged Gp17 (lane 7) proteins and submitted to EMSA analysis using a ISRE γ-³²P-labeled probe and native PAGE gel. In addition, extracts of IFN-α-treated cells were incubated with anti-STAT2 antibodies prior to incubation with γ-³²P-labeled probe (lane 3, *). (B) The same cell extracts were analyzed by immunoblotting with anti-pSTAT2 and anti-tubulin antibodies.