Rabies virus P protein interacts with STAT1 and inhibits interferon signal transduction pathways

Abstract

Rabies virus P protein is a cofactor of RNA polymerase. We investigated other potential roles of P (CVS strain) by searching for cellular partners using two-hybrid screening. We isolated a cDNA encoding the signal transducer and activator of transcription 1 (STAT1) that is a critical component of interferon type I (IFN-alpha/beta) and type II (IFN-gamma) signaling. We confirmed this interaction by glutathione S-transferase-pull-down assay. Deletion mutant analysis indicated that the carboxy-terminal part of P interacted with a region containing the DNA-binding domain and the coiled-coil domain of STAT1. The expression of P protein inhibits IFN-alpha- and IFN-gamma-induced transcriptional responses, thus impairing the IFN-induced antiviral state. Mechanistic studies indicate that P protein does not induce STAT1 degradation and does not interfere with STAT1 phosphorylation but prevents IFN-induced STAT1 nuclear accumulation. These results indicate that rabies P protein overcomes the antiviral response of the infected cells.

FIG. 1.

Mapping of P and STAT1 interacting domains. (A) Identification of the STAT1-binding domain on P by a two-hybrid system. Expression vectors encoding P or truncated P proteins fused to the DNA-binding domain of LexA were transformed into L40 yeast together with the construct encoding STAT1ΔN140 from Rattus norvegicus fused to the GAL4-activating domain. Cells were streaked onto plates containing medium lacking tryptophan and leucine (Trp⁻ Leu⁻) for double transformants or lacking tryptophan, leucine, and histidine (Trp⁻ Leu⁻ His⁻) to assay the activation of the HIS3 reporter gene. The induction of the LacZ reporter gene was assayed by the appearance of blue colonies as described in Material and Methods. +, Presence of blue yeast cells; −, no blue yeast cells. (B) Identification of the P-binding domain on STAT1. Expression vectors encoding various truncated STAT1 fused to the GAL4-activating domain were transformed into L40 cells together with the plasmid encoding the full-length P protein fused to the DNA-binding domain of LexA. Protein interactions were tested as described in panel A. (C) GST pull-down results. BSR cell extracts containing endogenous STAT1 were incubated with BL21 extracts containing GST alone, GST-P, GST-PΔC30, or GST-PΔC75 for 16 h at 4°C. Each mixture (input) was glutathione-agarose immobilized. Agarose-bound proteins were eluted three times (1, 2, 3) with PBS containing reduced glutathione and were analyzed by immunoblotting with anti-STAT1 antibody. Proteins were also stained with Coomassie brilliant blue to visualize the amount of agarose-immobilized GST proteins. WB, Western blot.

FIG. 2.

Expression of the rabies virus P protein inhibits IFN-α and IFN-γ signaling. (A) Human neuroblastoma SK-N-BE cells were transfected with ISRE-firefly luciferase reporter plasmid (pISRE-f.luc) and Renilla luciferase expression vector (pTK-r luc) and either empty vector or plasmid expressing P, PΔN52, or PΔC75 as indicated. At 24 h after transfection, cells were treated with 2,000 U/ml of human IFN-α (+) for 6 h prior to lysis and luciferase assays or left untreated (−). Neuroblastoma SK-N-BE cells were transfected with pISRE-f.luc and pTK-r luc and then infected (rabies) or not with 10 PFU/cell of rabies virus at 24 h after transfection. Cells were IFN treated at 48 h posttransfection as described above. 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. Bars which are statistically different from the corresponding control (P < 0.001) are labeled by asterisks. (B) Same as in panel A but using an IFN-γ-responsive GAS luciferase reporter gene instead of ISRE luciferase. (C) SK-N-BE cells were uninfected (−) or infected (+) with rabies virus (RV) for 24 h and were then treated with 2,000 U of human IFN-α/ml for 24 h. The expression of PKR and PML was studied by Western blot analysis with specific antibodies. (D) The same experiment was performed with 2,000 U/ml of human IFN-γ and the expression of IRF1 was analyzed by Western blot analysis..

FIG. 3.

Effect of P protein on antiviral response. (A) A control BSR clone and a clone of P-expressing BSR cells were treated with 2,000 U/ml IFN-α and then infected for various times as indicated with VSV at a MOI of 1 PFU/cell. Viral proteins were detected by Western blot analysis with a rabbit anti-VSV. +, IFN treated; −, not IFN treated. (B) Virus yields of the supernatant of cells infected for 7 h were obtained by plaque assay on BSR cells (expressed as PFU/ml).

FIG. 4.

Effect of rabies virus infection on STAT tyrosine phosphorylation and dimerization. SK-N-BE cells were infected with 10 PFU per cell of rabies virus (+, lanes 4, 5, and 6) or not infected (−, lanes 1, 2, and 3). Twenty hours after infection, cells were treated with 2,000 U/ml of human IFN-α for 10 min (lanes 2 and 5) or 20 min (lanes 3 and 6) or left untreated (−, lanes 1 and 4). (A) Cell lysates were analyzed directly by immunoblotting (SDS-12% PAGE) with anti-STAT1, anti-pSTAT1 (phosphorylated tyrosine 701), anti-STAT2, antitubulin, or anti-P antibodies. (B) Cell extracts were immunoprecipitated with anti-STAT2 antibody, and the immuncomplexes were then analyzed by immunoblotting with anti-STAT1 or anti-pSTAT1 antibodies. (C) Same as in panel A but using IFN-γ instead of IFN-α.

FIG. 5.

Rabies virus infection and P protein expression prevent IFN-α and IFN-γ induced STAT1 nuclear accumulation. (A) Human neuroblastoma SK-N-BE cell lines were uninfected or infected with rabies virus or transfected with plasmids expressing P or PΔC75 as indicated. Forty-eight hours after transfection or 24 h after infection, cells were unstimulated or stimulated with 2,000 U/ml of hIFN-α for 25 min and they were fixed, permeabilized, and then stained with anti-P and anti-STAT1 antibodies. (B) SK-N-BE cells were transfected with plasmids expressing P or PΔC75 or were infected with rabies virus and treated with 2,000 U/ml of hIFN-α (upper panel) or of hIFN-γ (lower panel). Cells were then stained with anti-P and anti-pSTAT1 antibodies. (C) SK-N-BE cells were transfected with plasmids expressing P and treated with 2,000 U/ml of hIFN-α or of hIFN-γ. Cells were then stained with anti-P and anti-pSTAT1 antibodies. The scale bars correspond to 40 μM.