Article Figures & Data
Figures
- FIG. 1.
STAT2 is a primary target of measles virus V protein. IFN-α/β and IFN-γ signaling interference by measles virus V (MeV) was tested using HEK293T (A) and 2fTGH (B) cells. Cells were transfected with an ISRE or GAS luciferase (Luc) reporter gene along with empty vector or measles virus V expression plasmid and were stimulated with IFN-α or IFN-γ (+) or left unstimulated (−) for 14 h prior to luciferase assays. (C) Analysis of STAT1 and STAT2 coprecipitation with measles virus V in 2fTGH cells [WT (2f)], U3A cells lacking STAT1 [U3A (−S1)], and U6A cells lacking STAT2 [U6A (−S2)]. STAT1 deficiency does not disrupt STAT2 interactions, but STAT2 deficiency disrupts STAT1 interactions. Complementation of STAT defects [(+S1), (+S2)] restores coprecipitation. C, control vector; V, measles virus V; IP, immunoprecipitation.
- FIG. 2.
Measles virus V CTD is necessary and sufficient to bind STAT2. (A) Schematic diagram of measles virus V (MeV) protein and variants. Numbers indicate amino acid positions; the shaded boxes represent the CTD. N, N terminal; C, C terminal. (B) Coimmunoprecipitation of endogenous STATs with V protein fragments. HEK293 T cells were transfected to express V proteins, cell extracts were prepared for immunoprecipitation (IP) with FLAG M2 affinity resin, and the eluates were evaluated by STAT immunoblotting. The left panel illustrates total lysate results; the right panel shows the results obtained after FLAG immunoprecipitation. (C) Details are similar to those described for panel B except that cells were transfected with GST-V protein constructs and lysates were precipitated with glutathione agarose prior to immunoblotting. Control mumps virus CTD (MuV C) did not bind STAT2. (D and E) Effects of GST fusion proteins on IFN-α/β signaling (D) and MDA5 signaling (E). HEK293 T cells were transfected with ISRE-luc or −110-IFN-β-luc reporter genes along with GST-V expression plasmids and MDA5 construct. Cells were left unstimulated (−) or were stimulated (+) with 1,000 U of IFN/ml (8 h) or subjected to transfection with 5 μg of poly:(IC)/ml (6 h) prior to luciferase assays. Bars illustrate average values (n = 3); standard deviations are indicated. (F) STAT1 interference maps to residues 110 to 130. Coimmunoprecipitation of V protein and variants with endogenous STAT1 in HEK293T cells was performed in an experiment similar to that described for panel B. (G) Effect of V protein variants on IFN-α/β signaling inhibition. A luciferase assay for IFN-α/β signaling was executed as described for panel D.
- FIG. 3.
CTD structure and amino acid sequence specify STAT2 interaction. (A) Comparison of measles virus and mumps virus V protein CTD sequences. Box1, Box2, and Box3 and zinc fingers 1 and 2 are indicated; five-pointed stars represent individual residues targeted for alanine substitution. (B) Coimmunoprecipitation (Co-IP) of box swap mutants Box1 (B1), Box2 (B2), and Box3 (B3), histidine substitution (H), or histidine/cysteine triple substitution (HC) with endogenous STAT2 in HEK293T cells. Vec, vector. (C) Effects of the V proteins on IFN-α/β signaling in HEK293T cells were tested as described for Fig. 1. (D) Effects of the box swap mutants on MDA5 signaling to the IFN-β reporter gene were tested as described for Fig. 2. (E) Effects of the V proteins on IFN-γ signaling in HEK293T cells were tested as described for Fig. 1.
- FIG. 4.
Point mutations disengage V-STAT2 association. (A) Sequence alignment of measles virus V protein with other paramyxovirus V proteins in the zinc finger region. Positions of Box1 and Box2 are shown; the residues indicated by five-pointed stars were replaced by the corresponding mumps virus V protein residues as described in the text. SV5, simian virus 5; HPIV2, type 2 human parainfluenza virus. (B) Coimmunoprecipitation (IP) of the point mutants with endogenous STAT2. (C) Effect of the mutants on IFN-α/β signaling. Dashed lines indicate 30% and 70% signaling interference thresholds. (D) Effect of the mutants in a IFN-γ signaling assay. (E) The measles virus V protein sequence was modeled on the SV5 structure (PDB accession no. 2B5L) by use of the Swiss PDB viewer available at http://www.expasy.org/spdbv/ (14). Each pair of images illustrates a ribbon view on the left and a surface view on the right, with front and back faces indicated by 180° rotation. Coloration corresponds to the activity level of mutations for each position. Gray represents nonmutated residues, cyan indicates neutral substitutions, yellow represents mutations that retain 30 to 80% activity, and red represents mutations that are more than 70% defective.
- FIG. 5.
D248F mutation prevents V-mediated STAT2 cytoplasmic sequestration. 2fTGH cells were transiently transfected with empty vector or expression plasmids for the WT or mutant measles virus V (MeV) proteins indicated. Cells were left unstimulated (UNT) or were treated with 1,000 U of IFN-α/ml for 30 min prior to fixation, permeabilization, and sequential staining for a FLAG epitope tag to detect V protein and antiserum to detect endogenous STAT2. Arrows point to V-expressing cells.
- FIG. 6.
D248F mutation is required for antiviral evasion. (A) Reporter virus assay for antiviral effects. 2fTGH stable cell lines expressing the empty vector or the V proteins indicated were left untreated (−IFN) or treated with IFN-α (+IFN) for 8 h and then infected with 9 × 105 PFU of VSV-GFP/well for 22 h prior to fluorescence microscopy. (B) Demonstration of V protein expression in stable cell lines with FLAG antibody and anti-α-tubulin control. (C) Quantification of VSV-GFP. The cells described for panel A were harvested, and 10,000 cells were analyzed by flow cytometry for GFP fluorescence.
