Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade
Figure 4
The C-terminal RNR R1 homology domain of M45 is required for the inhibition of NF-κB activation and interaction with NEMO.
(A) Schematic representation of M45 truncation mutants used in this and a previous [36] study. The C-terminal RNR R1 homology domain is shown in white, the unique N terminus in grey. The RHIM is marked black. (B) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting. (C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies. (D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).