Extended Data Fig. 2: TRIM5α is a VACV restriction factor.
From: TRIM5α restricts poxviruses and is antagonized by CypA and the viral protein C6
a) Immunoblot showing TRIM5 levels in T-REx-293 (WT) and derivative TRIM5-/- (KO1/2) cells. b) Immunoblot showing TRIM5 levels in HeLa (WT) and derivative TRIM5-/- (KO1/2) cells. c) Infectious VACV titres following infection of cells described in b) at 5 PFU/cell for 16 h. n = 3/condition. d) Infectious VACV titres following infection of cells described in a) at 0.01 PFU/cell for 24 h. n = 3/condition. e) Plaque image (left) and plaque area quantification (right) 24 hpi of cells indicated in c) with VACV A5-GFP. n ≥ 6/condition. f) Infectious VACV titres following infection of cells described in b) at 0.01 PFU/cell for 24 h. n = 3/condition. g) Immunoblot showing inducible expression (+Dox) of FLAG-tagged TRIM5α/γ/δ isoforms, in T-REx-293 cells. h) Plaque image 24 hpi of cells indicated in g) with VACV A5-GFP. i) Plaque area quantification of h). n ≥ 48/condition. j) Infectious VACV titres following infection of cells described in g) at 0.01 PFU/cell for 24 h. n = 3/condition. k) Infectious VACV titres following infection of cells described in g) at 5 PFU/cell for 16 h. n = 3/condition. l) Immunoblot showing inducible expression (+Dox) of TAP-tagged TRIM5α/γ/δ isoforms, introduced into T-REx-293 TRIM5-/- cells. m) Plaque image 24 hpi of cells indicated in l) with VACV A5-GFP. n) Infectious VACV titres following infection of cells described in l) at 0.01 PFU/cell for 24 h. n = 3/condition. o) Immunoblot showing inducible expression (+Dox) of TAP-tagged TRIM5α mutants, L19R, N70A, R119E and ΔSPRY introduced into T-REx-293 TRIM5-/- cells. p) Plaque images of VACV A5-GFP infection of cells described in o) for 24 h. q) Infectious VACV titres following infection of cells described in o) at 0.01 PFU/cell for 24 h. Viral titres were normalised to the respective uninduced (-dox) controls. n = 3/condition. In g-n) cells were treated with 150 ng/mL doxycycline from seeding for 24 h to express tagged proteins before harvest or infection. In o-q) doxycycline was added at 24 h after seeding. In a), b), g), l) and o) protein extracts were analysed by SDS-PAGE and immunoblotted for the indicated epitope/protein. Data shown in d), h), i), m) and p) are representative of three independent experiments, and in b), e), f), j), k) and q) are from two. Data from b) and d-f) were analysed using One-Way Welch’s ANOVA test. Data from i-k), n) and q) were analysed using two-tailed unpaired Student’s t-test. Analyses were performed on GraphPad Prism. Mean ± s.e.m.
