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. Author manuscript; available in PMC: 2010 Oct 1.
Published in final edited form as: Cell. 2010 Apr 16;141(2):315–330. doi: 10.1016/j.cell.2010.03.029

Figure 2. K63 polyubiquitination is Essential for RIG-I Activation.

Figure 2

(A) Ubc5 and Ubc13/Uev1a activate RIG-I in vitro. RIG-I was incubated with E1, different E2s as indicated, TRIM25, ubiquitin, RNA and ATP, followed by IRF3 dimerization assay as described in Figure 1D. The E2 proteins (2 μg) were analyzed by Coomassie Blue staining (lower panel). (B) Ubc5 and Ubc13 are required for viral activation of MAVS in the mitochondria. U2OS cells stably integrated with tetracycline-inducible shRNA against Ubc5b/c and Ubc13 were treated with or without tetracycline (Tet). After viral infection for the indicated time, mitochondrial fraction (P5) was prepared and the MAVS activity was measured by IRF3 dimerization assay. (C) K63 of ubiquitin is essential for RIG-I activation in vitro. RIG-I and IRF3 activation assays were performed using Ubc5c (upper panel) or Ubc13/Uev1a (lower panel) as the E2, and various ubiquitin mutants as indicated. KO, lysine-less mutant; MeUb, methylated ubiquitin. (D) K63 polyubiquitination is essential for viral activation of MAVS in the mitochondria. U2OS cells stably integrated with tetracycline-inducible shRNA against endogenous ubiquitin genes and a rescue expression vector for wild-type or K63R mutant of ubiquitin were grown in the presence or absence of tetracycline. After infection by Sendai virus, mitochondria (P5) were prepared to measure MAVS activation. The expression of MAVS and ubiquitin was analyzed by immunoblotting (lower panels). The HA antibody detects HA-Ub(WT) or HA-Ub(K63R) expressed from the transgene rescue vector. See also Figure S2.