Murine Gamma Herpesvirus 68 Hijacks MAVS and IKKβ to Abrogate NFκB Activation and Antiviral Cytokine Production
Figure 5
γHV68 infection induces RelA degradation in an IκBα-independent manner.
Mouse embryonic fibroblasts (MEFs) were infected with γHV68 K3/GFP at an MOI of 20 (A, C, and E), or vesicular stomatitis virus (VSV, MOI = 10) (D), or Sendai virus (SeV, 4000 HA units) (D), or treated with lipopolysaccharides (LPS, 200 µg/ml). (A) Protein levels of RelA, IκBα, and β-actin in whole cell lysates (WCLs) from γHV68-infected wild-type MEFs at 4 h.p.i. were analyzed by immunoblot. MG, MG132 (20 µM); Ch, chloroquine (50 µM). (B and C) Wild-type MEFs stably expressing the Flag-tagged IκBα super-suppressor (IκBαΔN) were established as described in Materials and Methods. The expression of endogenous and exogenous IκBα was examined by immunoblot (B). WCLs of γHV68-infected control (Vector) or IκBαΔN-expressing MEFs were separately analyzed by immunoblot with indicated antibodies (C), to facilitate the detection of RelA that was reduced by IκBαΔN expression. Relative quantity of RelA in γHV68-infected MEFs was normalized to β-actin (right). See also Figure S10. (D) Wild-type MEFs were infected with VSV, SeV, or treated with LPS, and WCLs were analyzed by immunoblot with indicated antibodies. (E) MG132 treatment induced RelA accumulation in the cytosol of γHV68-infected MEFs. Wild-type MEFs were treated with TNFα (10 ng/ml, 30 min) (left) or infected with γHV68 without or with MG132 (20 mM) for 2 hours (right). A representative optical field is presented for each group. Data represent three independent experiments.