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. 2006 Aug;80(15):7555-68.
doi: 10.1128/JVI.02720-05.

Rinderpest virus blocks type I and type II interferon action: role of structural and nonstructural proteins

Sambit K Nanda  1 , Michael D Baron
Affiliations

Rinderpest virus blocks type I and type II interferon action: role of structural and nonstructural proteins

Sambit K Nanda et al. J Virol. 2006 Aug.

Abstract

Rinderpest virus (RPV) is a paramyxovirus closely related to the human pathogen Measles virus. It causes severe disease in cattle, buffalo, and some wild animals; although it can infect humans, it does not cause disease. Here, we demonstrate that RPV blocks the action of both type I (alpha) and type II (gamma) interferons (IFNs) by blocking the phosphorylation and nuclear translocation of STAT1 and STAT2 and that this block is not related to species specificity. In addition, both wild-type virulent and vaccine strains of the virus blocked IFN action. Unlike the case with some other paramyxoviruses, neither STAT1 nor STAT2 is degraded upon virus infection. STAT1 is bound by both the viral structural protein P, and thereby recruited to concentrations of viral protein in the cell, and the nonstructural protein V. Although both P and V proteins bind to STAT1 and can block IFN action when expressed in transfected cells, the IFN antagonist activity of the P protein is weaker than that of the V protein. The viral C protein also seems to weakly block IFN-induced activation of STAT1 in transfection experiments. However, studies with knockout viruses showed that the viral V protein appears to be the dominant inhibitor of IFN signaling in the context of virus infection, since prevention of viral V expression restored the IFN sensitivity of infected cells. Although a change in the distribution pattern of STAT2 was observed in virus-infected cells, STAT2 was not bound by any viral protein.

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Figures

FIG. 1.
FIG. 1.
RPV infection inhibits the tyrosine phosphorylation of STAT1 and STAT2 in response to IFN-α and IFN-γ. BSF cells (a, d), A549 cells (b, e), or Vero cells (c, f) were infected with RPV Saudi/81 at an MOI of 5 or left uninfected. Eighteen hours postinfection, the cells were treated with or without IFN-α (a to c), bovine IFN-γ (d) or human IFN-γ (e to f) for 30 min, and the cells were harvested and the levels of STATs and tyrosine-phosphorylated STAT proteins determined by Western blot analysis with the corresponding specific antibodies, as indicated to the left of each panel. PCNA levels served as a recovery/loading control; the levels of PCNA were checked for every experiment, although only representative data sets are presented here. Samples were also probed for the presence of viral protein (P) to confirm infection (a to c). The figure shows results from one of three experiments which all yielded consistent results.
FIG. 2.
FIG. 2.
RPV infection blocks the expression of type I or type II IFN-inducible proteins. (a) BSF and A549 cells were mock infected or infected with RPV Saudi/81 at an MOI of 5 as indicated on the figure; 24 h postinfection the cells were treated with IFN-α overnight or left untreated, and the level of MxA protein was determined by Western blotting. PCNA acted as the loading control. (b) BSF and A549 cells were infected as in the legend for panel a; 24 h postinfection, the cells were treated with bovine or human IFN-γ as appropriate or left untreated. After a further 18 h incubation, the cells were harvested and the levels of IRF-1 were determined by Western blotting. The figure shows results from one of two consistent experiments.
FIG. 3.
FIG. 3.
RPV virus infection alters STAT protein distribution in the cell. (a) BSF cells were infected with RPV Saudi/81 at an MOI of 0.1 for 18 h. The cells were fixed and stained with monoclonal antibody against either viral phosphoprotein (P) or nucleocapsid protein (N) and rabbit polyclonal antibody against either STAT1 or STAT2. The viral N or P was visualized using Alexa Fluor 568 (red) and the STAT1 or STAT2 using Alexa Fluor 488 (green) secondary antibodies. Nuclei were counterstained with DAPI (blue). (b) BSF cells were infected with RBOK, RPV V−, RPV C−, or RPV V− C− at an MOI of 0.1 for 18 h, as indicated on the left hand side of each panel. Cells were fixed and stained with monoclonal antibody against viral phosphoprotein (P; red) and rabbit polyclonal antibody against STAT1 (green); DAPI was used to stain nuclei (blue). Images were acquired by sequential laser scanning on the confocal microscope. For all the images, the left-hand panel represents merged images of the nucleus (blue) and viral protein (red) showing the infected cells, the middle panel shows the specific STAT1 or STAT2 protein stain, and the right-hand panel is an overlay of all three colors.
FIG. 4.
FIG. 4.
STAT1 is bound to viral P, W, and V proteins. Vero cells were transfected with either empty plasmid vector or plasmid encoding the RPV P (pcDNA-PCSTOP), N (pcDNA-N), L (with an amino-terminal HA tag) (pcDNA-HAL), V (with an amino-terminal c-Myc tag) (pcDNA-c-Myc-V), W (with carboxy-terminal V5 tag (pcDNA-W-V5), or C (pcDNA-C-V5), as indicated at the top of the figure. Forty-eight hours posttransfection, the transfected cells were lysed and the lysates were immunoextracted with mouse anti-P, rabbit anti-N, rat anti-HA, rabbit anti-c-Myc, or mouse anti-V5 antibodies. Cells transfected with empty vector were lysed and STAT1 or STAT2 was precipitated with the respective rabbit polyclonal antibody. The immunoprecipitated materials were probed by Western blotting for the presence of STAT1 (top panel) or STAT2 (middle panel). Rabbit polyclonal antibody against STAT1 was used to detect STAT1 in all the immunoprecipitated material. The primary antibody was detected with TrueBlot anti-rabbit immunoglobulin G (precipitates with anti-STAT1, anti-P, and anti-N) or HRP-antirabbit (precipitates with anti-HA, anti-c-Myc, or anti-V5). Mouse monoclonal anti-STAT2 antibody was used to detect STAT2 in the immune precipitates with polyclonal anti-N or anti-c-Myc antibodies, while polyclonal rabbit-anti-STAT2 was used to detect STAT2 in immunoprecipitates with mouse anti-P, mouse anti-V5, or rat anti-HA antibodies. The bottom panels show that the immunoprecipitates contained the expected viral protein, as shown by probing with rabbit anti-P, mouse anti-N, rat anti-HA, mouse anti-P/V, or mouse anti-V5, respectively. Note that samples precipitated with anti-V5 and probed with the same antibody showed clear immunoglobulin G-derived bands (the light chain visible in the blot showing C protein expression). i.p., immunoprecipitation; w.b., Western blotting.
FIG. 5.
FIG. 5.
RPV V, P, and C all inhibit IFN-induced phosphorylation of STATs. BSF cells were transfected with plasmids expressing GFP or N-, V-, P-, C-, or C-V5-tagged proteins as indicated. Twenty hours posttransfection, STAT1/STAT2 phosphorylation was induced where indicated by treatment with 1,000 U/ml of IFN-αA for 30 min. The cells were fixed/permeabilized with methanol:acetone and stained with either the relevant rabbit polyclonal antibody to the expressed protein and mouse monoclonal antibody against pY701-STAT1 (a to r), mouse monoclonal antibody recognizing both P and V proteins and rabbit polyclonal (s to dd), or mouse monoclonal anti-V5 tag and rabbit anti-pY689-STAT2 (ee to jj). Expressed proteins were detected using Alexa Fluor 568 (red) except for GFP, which was detected with Alexa Fluor 488 (green). STAT1 and STAT2 were detected using Alexa Fluor 488 (green) for all except for cells transfected with plasmid expressing GFP, when Alexa Fluor 568 (red) was used. Nuclei were stained with DAPI (blue). All images were obtained by sequential laser scanning with the confocal microscope. For all the images, the top panel shows the overlay of nuclear staining (DAPI) and the expressed protein, the middle panel shows the phosphorylated STAT, and the bottom panel shows the overlay of phosphorylated STATs and expressed (exp.) protein.
FIG. 6.
FIG. 6.
RPV V, P, and C all inhibit IFN-γ-induced phosphorylation of STAT1. BSF cells were transfected with plasmid expressing GFP or N, V, P, or C proteins (as indicated at the bottom of the figure) for 20 hours. STAT1 phosphorylation was induced by treatment with 5 ng/ml of bovine IFN-γ for 30 min. The cells were fixed/permeabilized with methanol:acetone and stained with the relevant rabbit polyclonal antibody to the expressed protein and mouse monoclonal antibody against pY701-STAT1. Expressed proteins were detected using Alexa Fluor 568 (red) except for GFP, which was detected with Alexa Fluor 488 (green). Phosphorylated STAT1 was detected using Alexa Fluor 488 (green) for all except cells transfected with plasmid expressing GFP, when Alexa Fluor 568 (red) was used. Nuclei were stained with DAPI (blue). All images were obtained by sequential laser scanning with the confocal microscope. For all the figures, the bottom panels show the overlay of phosphorylated STAT1 and expressed protein. The top panel shows the overlay of nuclear staining (DAPI) and the expressed protein.
FIG. 7.
FIG. 7.
RPV V and P inhibit the induction of expression from IFN-responsive promoters. Vero cells were transfected with plasmids encoding the RPV P, V, or C proteins or blank vector, along with pJATLacZ and (a) pISRE-luc or (b) pGAS-luc. After 24 h of transfection, the cells were treated for 18 h with (a) IFN-α or (b) IFN-γ, lysed, and the luciferase and β-galactosidase activities measured. The ratio of these two activities was taken as the relative luciferase activity (in RLU). Shown are the data from a representative experiment; error bars represent one standard deviation. The induction (n-fold) (RLU + IFN/RLU − IFN) is shown for each −IFN/+IFN pair.
FIG. 8.
FIG. 8.
Effect of RPV knockout virus on IFN-induced STAT1 activation. BSF cells were infected with the indicated virus (shown on the left of the panels) at an MOI of 0.1 for 18 h. The cells were treated with either 1,000 IU/ml of IFN-αA or 5 ng/ml of bovine IFN-γ, or left untreated, prior to fixation/permeabilization using methanol:acetone (1:1). The cells were stained with rabbit anti-P antibody to detect infected cells and mouse anti-pY701-STAT1 antibody followed by Alexa Fluor 568 (red) antirabbit plus Alexa Fluor 488 (green) antimouse. The nuclei were counterstained with DAPI (blue). All images were obtained by sequential laser scanning with the confocal microscope. For all combinations, the left panels show the overlay of DAPI and P protein staining, the middle panels show phosphorylated STAT1, and the right panels show the overlay of STAT1P and P protein staining.
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