Fig. 2
From: Primate TRIM34 is a broadly-acting, TRIM5-dependent lentiviral restriction factor
TRIM34 requires TRIM5α to restrict SIVAGM-SAB. a–b. THP-1 TRIM34 clonal KO cells containing doxycycline-inducible TRIM34 from humans (a) or rhesus macaques (b) were transduced with lentiviral vectors encoding Cas9 and 1 of 2 independent sgRNA against TRIM5 or a non-targeting control to generate pooled TRIM5 knockout or NTC cell lines. Pooled KO efficiency was assessed by ICE analysis (Additional file 1: Chromatograms S2–S5). For human TRIM34-expressing cell lines, ICE KO efficiency scores were as follows: TRIM5 sgRNA 1 (triangles) = 76%, TRIM5 sgRNA 2 (circles) = 90%. For rhesus macaque TRIM34-expressing cells, ICE KO scores were as follows: TRIM5 sgRNA 1 (triangles) = 83%, TRIM5 sgRNA 2 (circles) = 93%. Thus, there existed 4 different experimental conditions: no TRIM34 or TRIM5α expression (black symbols), only endogenous TRIM5α expression (blue symbols), only overexpressed TRIM34 (green symbols), both endogenous TRIM5α and overexpressed TRIM34 (orange symbols). 1 day after doxycycline induction, cells were infected with SIVAGM-SAB CA particles. 2 dpi, infectivity was quantified by flow cytometry. Relative infectivity is normalized to mean infectivity in TRIM5KO, TRIM34-uninduced cells (black symbols). Infection of each cell line with each virus was performed a total of 6 times across 2 different occasions. Combined data are represented as mean +/- s.d., where each point represents a unique infection. Fold inhibition is indicated where applicable. c. HeLa cells were transduced to stably express primate TRIMΔSPRY orthologues or empty vector control. Expression was visualized by immunoblotting. d. HeLa cells stably expressing primate TRIM34ΔSPRY orthologues, human TRIM5ΔSPRY, or empty vector control were infected with N-MLV (grey bars) or NB-MLV (white bars). Level of infection was quantified 2 dpi by flow cytometry. Infection of each cell line with each virus was performed a total of 6 times across at least 2 different occasions. Combined data are represented as mean +/- s.d., where each point represents a unique infection. Fold rescue relative to empty vector control cells is indicated where applicable. a–b, d. p values were calculated by Brown-Forsythe and Welch’s 1-way ANOVA with Dunnett’s T3 test for multiple comparisons. ns nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001