Extended Data Fig. 5: L3 dimerisation.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a) L3 dimerisation is stabilised by TRIM5α and reversed by CypA WT. T-REx-293 TRIM5-/-CypA-/- cells were co-transfected with TAP-tagged L3 or N1, HA-tagged L3, TRIM5α WT, ZAP-L, CypA WT, R55A or F113A with or without 5 µM CsA for 24 h. b) Relative band intensity of HA-tagged L3 in a) AP samples. Graph shows band intensity of HA-tagged L3 from three independent experiments, normalised to TAP-tagged L3 and relative to the sample transfected with TAP-tagged and HA-tagged L3. n/condition is as indicated. Data points were excluded in samples where n < 3 due to high background noise causing inaccuracy in quantification. c) Stabilisation of L3 dimerisation by TRIM5α requires its E3 ubiquitin ligase activity. T-REx-293 TRIM5-/-CypA-/- cells were co-transfected with TAP-tagged L3 or N1 and HA-tagged L3, TRIM5α WT, L19R or CypA WT. d) Relative band intensity of HA-tagged L3 in c) AP samples. Graph shows band intensity of HA-tagged L3 from three independent experiments, normalised to TAP-tagged L3 and relative to the sample transfected with TAP-tagged and HA-tagged L3. n = 3/condition. Data were analysed using were analysed using One-Way Welch’s ANOVA test (p = 0.0007) and pairwise comparisons were performed using Dunnett’s T3 multiple comparisons test on GraphPad Prism. Mean ± s.e.m. e) Modification of L3 by TRIM5α WT requires its E3 ubiquitin ligase activity and is reversed by CypA WT. T-REx-293 TRIM5-/-CypA-/- cells were co-transfected with TAP-tagged TRIM5α WT, L19R or CypA WT and HA-tagged L3 or CypA. Tagged viral proteins were affinity-purified using Strep-Tactin beads. Inputs and AP proteins in a), c) and e) were analysed by SDS-PAGE and immunoblotted for the indicated epitope/protein. Data shown are representative of three independent experiments.
