Figure 1.
pM27 is essential and sufficient to reduce the amount of human and mouse STAT2.
(A) Human MRC-5 and mouse M2-10B4 cells were infected (0.3 or 3 PFU/cell, 16 h) with indicated VACVs, lysed and subjected to western blotting. (B) STAT2-HA expressing cells were infected as in (B) for 24 h. Figure 1B is part of a larger experiment which is shown in Figure S6. (C) Cells of indicated genotype were infected (5 PFU/cell, 15 h) with wt-VACV or M27-Flag-VACV, lysed and analyzed by western blotting. Please note cross reactivity of the STAT3 antibody for STAT1.
Figure 2.
pM27 affects STAT2 protein levels.
(A) STAT2-HA cells were infected with indicated MCMV mutants (30 PFU/cell). Cells were incubated with or without cycloheximide (100 µg/ml) and actinomycin D (5 µg/ml). After 1, 2 or 5 h cells were analyzed by western blotting. The inhibitors precluded expression of pp89-IE1 and pM27-HA indicating tight inhibition. (B) 35S-L-Cys/L-Met labelled STAT2-HA cells were infected (5 PFU/cell, 20 h). Cells were metabolically labelled (30 min) and subsequently chased for the indicated time. STAT2-HA was precipitated using an HA-specific antibody. Proteins were separated by 8% SDS-PAGE and visualized by autoradiography. (C) As in (B), but cells were infected (20 PFU/cell) for 90 min prior to the indicated pulse-chase regime. Asterisks indicate non-specifically precipitated proteins.
Figure 3.
pM27 facilitates the proteasome to degrade STAT2.
(A) STAT2-HA cells were infected with VACV (3 PFU/cell, 8 h) expressing full-length M27-Flag or a non-functional (M27-Flag265–682) truncation mutant and treated for 4 or 6 h with 10 µM MG132 or solvent DMSO and analyzed by western blotting. (B) STAT2-HA and STAT2−/− cells were infected (3 PFU/cell, 4 h) with VACV expressing pM27-Flag or a non-functional mutant (M27-Flag1–487), treated (4 h) with 5 or 10 µM lactacystin or DMSO and analyzed by western blotting. * indicates an unspecifically detected protein running close to pM27-Flag. (C) STAT2-HA cells were infected (20 PFU/cell, 2 h) with ΔM27-MCMV or M27-HA-MCMV and incubated (3 h) with CHX (50 µg/ml) alone or a combined treatment of CHX, lactacystin (10 µM) and MG132 (20 µM). (D) STAT2-HA cells were infected (10 PFU/cell) with M27-Flag-VACV or wt-VACV. 4 h post infection MG132 (10 µM) was added. 24 h post infection (M27-Flag-VACV) or 20 h post infection (wt-VACV) cells were lysed and STAT2-HA was precipitated by HA-specific antibody. Precipitated proteins were separated by SDS-PAGE and blotted. The membrane was probed with a Ub-specific and reprobed with an HA-specific antibody.
Figure 4.
pM27 co-precipitates a ∼125 kDa protein.
(A) STAT2-HA (2HA) and STAT2-deficient (2−/−) cells were infected (5 PFU/cell, 24 h) with wt-MCMV, ΔM27-MCMV, M27-HA-MCMV or left uninfected. Cells were starved (1 h) and then 35S-L-Met/L-Cys labelled (150 min). Lysates were subjected to IP using an HA-specific antibody. The asterisk indicates a non-specific protein. (B) As in (A), but cells were infected (10 PFU/cell, 48 h) with M27-HA-MCMV, M28-HA-MCMV, wt-MCMV or left uninfected. Lysates were split and analyzed by western blotting in parallel to autoradiography. pM28-HA forms multimers leading to the weak high-molecular weight signal in the rightmost lane. (C) As in (A), but cells were infected for 22 h. SDS-PAGE was followed by Coomassie-staining. (D) STAT2-HA or STAT2−/− cells were infected (5 PFU/cell, 20 h) with M27-Flag-VACV or wt-VACV, lysed and analyzed by HA-specific IP. Proteins were separated by SDS-PAGE and visualised by silver staining.
Figure 5.
pM27 co-precipitates with DDB1.
(A) STAT2-HA and STAT2−/− cells were infected (5 PFU/cell, 24 h) with indicated MCMV-mutants (wt-MCMV, M27-HA-MCMV or M28-HA-MCMV), lysed and subjected to IP using HA-specific antibody, an isotype or no antibody. Precipitated proteins were analyzed by western blotting. (B) Infection was done as in (A). Cells were lysed and used for HA-specific IP. Precipitated proteins were analyzed by western blotting and by silver staining. (C) STAT2-HA and STAT2−/− cells were infected (5 PFU/cell, 18 h) with M27-Flag-VACV, incubated with MG132 (10 µM, for the last 4 h), lysed and subjected to IP using a Flag-specific antibody or an isotype antibody. Precipitated proteins were detected by western blotting. (D) MRC-5 cells were infected (5 PFU/cell, 18 h) with M27-Flag-VACV, lysed and analyzed by IP with Flag-specific antibodies. The immune complexes were separated by SDS-PAGE and analyzed by western blotting.
Figure 6.
Truncation analysis of pM27-Flag indicates correlation between DDB1 binding and STAT2 degradation.
(A) The schema depicts the generated M27 truncation mutants, all constructs are Flag-epitope tagged and expressed by recombinant VACVs. ‘27HR’ indicates a conserved domain shared between homologous proteins of related cytomegaloviruses. (B) STAT2-HA cells were infected with the indicated pM27-expressing VACV (16 h; 5 PFU/cell). Proteins from cell lysates were immunoprecipitated using a Flag-specific antibody (upper panel). The precipitated proteins were detected by western blotting. A part of the lysate was acetone precipitated and used to analyse the overall protein amounts (lower panel). (C) Cells were infected with wt-VACV or a VACV expressing a pM27-Flag protein, lacking aa 5–118 at the N-terminus and 652–682 at the C-terminus. After precipitation with α-Flag antibody, proteins were analyzed by western blotting. (D) pIRES-EGFP plasmids expressing the indicated pM27-Flag mutants were transiently transfected into human HeLa cells and subjected to α-Flag immunoprecipitation and subsequent western blotting using DDB1-specific and Flag-specific antibodies.
Figure 7.
siRNA mediated knock-down of DDB1 restores STAT2 amount and phenocopies M27-deficiency.
(A) Primary IFNAR1-deficient MEF cells (passage 3) were transfected with 200 nM DDB1-siRNA, M27-siRNA and an irrelevant siRNA, respectively. 24 h later cells were infected for 4 or 24 h with 10 PFU/cell M27-HA-MCMV or ΔM27-MCMV. Cells were lysed and subjected to western blotting. (B) Primary MEFs were transfected as in (B), infected (0.1 PFU/cell, 48 h) with Δm157-MCMV:luc and luciferase activity was measured. (C) As in (C) but cells were infected with wt-MCMV or ΔM27-MCMV and infectious virus progeny was determined by plaque titration.
Figure 8.
Current model of pM27 function.
pM27 bridges STAT2 to DDB1-containing ubiquitin-ligase complexes thereby inducing poly-ubiquitination and subsequent proteasomal degradation of STAT2 to antagonize induction of interferon stimulated genes.