Extended Data Fig. 6: Localisation of L3 and activation of innate immunity.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a) TAP-tagged TRIM5α and HA-tagged L3 localisation during vΔC6 infection. T-REx-293 TRIM5-/-CypA-/- cells were transfected with TAP-tagged TRIM5α and HA-tagged L3 and infected with vΔC6 at 2.5 PFU/cell for 10 h. Cells were stained with DAPI (blue) and antibodies against FLAG (red) and HA (green). White arrows indicate viral factories. Images were acquired using a Zeiss LSM 700 confocal microscope. b) TRIM5α localisation during vL3Li +/- IPTG. T-REx-293 TRIM5-/-CypA-/- cells were transfected with TAP-tagged TRIM5α for 12 h. Cells were infected with vL3Li, whose L3 expression is induced only upon IPTG treatment, at 2.5 PFU/cell for 10 h. Cells were stained with DAPI (blue), and antibodies against FLAG (red). Images were acquired using Zeiss LSM 700. Quantitative analysis shown in Fig. 4f. c) IκBα degradation following TAP-tagged L3 expression. T-REx-293 TRIM5-/-CypA-/- cells stably transfected with TAP-tagged EV or L3 were serum starved for 4 h prior to doxycycline treatment for the indicated times. TNF-α stimulation was for 30 min. d) Fold induction of IL6, CXCL8, CCL2, NFKBIA and CXCL10 mRNAs. T-REx-293 TRIM5-/-CypA-/- cells stably transfected with TAP-tagged EV or L3 were induced with doxycycline for 4 h before harvest and analysis by RT-qPCR. n = 3/condition. e) L3 and TRIM5α WT or mutant N70A stimulate NF-κB synergistically. TRIM5-/-CypA-/- cells were co-transfected with 100 ng NF-κB-luciferase and 10 ng Renilla luciferase alongside either EV, L3, TRIM5α WT or mutants L19R, N70A and ΔSPRY, individually, or TRIM5α WT or mutants with L3 or C1. Cells were harvested 24 h after transfection, firefly luciferase activity was measured and normalised to Renilla luciferase. Fold induction is relative to EV. n = 3/condition. Data shown in a), b), and e) are representative of three independent experiments and c) and d) are from two. Data from d) was analysed using two-tailed unpaired Student’s t-test on GraphPad Prism. Data from e) was analysed using One-Way Welch’s ANOVA test (p < 0.0001) and pairwise comparisons were performed using Dunnett’s T3 multiple comparisons test. Mean ± s.e.m.
