Extended Data Fig. 1: VACV protein C6 induces proteosomal degradation of TRIM5α.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a) Immunoblots showing TRIM5α abundance in T-REx-293 TRIM5-/- cells complemented with inducible expression (+dox) of TAP-tagged TRIM5α and infected with WT or vΔC6. b) C6 and TRIM5α co-precipitate. HEK293T cell lines were induced (+Dox) to express TAP-tagged C6 or B14 +/- MG132. c) C6 and TRIM5α interact. HA-tagged TRIM5, HDAC1 and HDAC5 were co-expressed with TAP-tagged C6 using the wheat germ transcription/translation system. HDAC1 and HDAC5 were used as negative and positive controls for direct interaction with C6, respectively. d) C6-mediated ubiquitylation of TRIM5α. T-REx-293 TRIM5-/-CypA-/- cells were transfected to co-express TAP-tagged B14 or C6, FLAG-tagged WT/K48/K63 ubiquitin and HA-tagged TRIM5α N70A. Red bracket indicates modified TRIM5α N70A. Graph shows band intensity of higher molecular mass TRIM5α N70A indicated by the red bracket from three independent experiments, normalised to the unmodified TRIM5α N70A band shown by the HA lower exposure blot and relative to the sample transfected with TAP-tagged C6, FLAG-tagged WT ubiquitin and HA-tagged TRIM5α N70A. n = 3/condition. Data was analysed using One-Way Welch’s ANOVA test (p = 0.0031) and pairwise comparisons was performed using Dunnett’s T3 multiple comparisons test. Mean ± s.e.m. Protein extracts in a) and inputs and AP proteins in b-d) were analysed by SDS-PAGE and immunoblotted for the indicated epitope/protein. Data shown in a), b) and d) are representative of three independent experiments and c) is from two.
