Fig. 4: Identification of TRIM5α and CypA interaction partners.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a–c, Volcano plots showing proteins co-purifying with TAP–TRIM5α (a), CypA (b) or CypA plus 20 μM CsA (c) and analysed by mass spectrometry. T-REx-293 TRIM5−/−CypA−/− cells were transfected with TAP–TRIM5α or CypA and infected with vΔC6 at 3 PFU per cell for 10 h. Dashed lines indicate a false discovery rate of <0.05. The P values for L3 in a–c are 0.01763, 0.12212 and 0.93991, respectively. d, L3 precipitation with TRIM5α and CypA. T-REx-293 TRIM5−/−CypA−/− cells were transfected with HA–L3 and TAP–TRIM5α or CypA ± CsA and infected with vΔC6 at 3 PFU per cell for 12 h, or mock-infected. e, Mapping of the L3 interaction domain. T-REx-293 TRIM5−/−CypA−/− cells were co-transfected with HA–L3, HA–C6 or HA–N1 and TAP–TRIM5α WT and mutants (Fig. 1h). IP, Immunoprecipitation. f, TRIM5α localization for vL3Li ± IPTG. T-REx-293 TRIM5−/−CypA−/− cells were transfected with TAP–TRIM5α for 12 h. Cells were infected with vL3Li at 2.5 PFU per cell for 10 h. The graph shows the relative number of cells in which TRIM5α is either localized in the viral factory only or also elsewhere in the cytoplasm ± IPTG. Data are from three independent experiments. n ≥ 36 per experiment. g, TRIM5α and L3 stimulate NF-κB activation. T-REx-293 TRIM5−/−CypA−/− cells were co-transfected with NF-κB–luciferase (Luc) and Renilla luciferase reporters alongside either empty vector, TRIM5α, L3 or CypA WT individually, or TRIM5α and L3 with CypA WT or R55A, or L3 with CypA WT or R55A. After 20 h, firefly luciferase activity was measured and normalized to Renilla luciferase. Fold induction is relative to empty vector. n = 3 per condition. TAP-tagged (d) or HA-tagged (e) proteins were precipitated using Strep-Tactin and anti-HA agarose beads and analysed alongside inputs by immunoblotting. Data shown are representative of three independent experiments, except a–c. In a–c, enriched proteins in the TRIM5 and CypA pulldowns were identified by comparison with empty vector using a two-sided Student’s t-test and a permutation-based false discovery rate of <0.05. Data from f,g were analysed using one-way Welch’s ANOVA test (where P < 0.0001 in f and P < 0.0001 in g), and pairwise comparisons were performed using post-hoc Dunnett’s T3 multiple comparisons test. Data are mean ± s.e.m. (f,g).
