Fig. 3

TRIM5α v1 loop is necessary for restriction of SIV_AGM-SAB. a. THP-1 TRIM34 clonal KO cells were electroporated with multiplexed sgRNA against TRIM5. Single cell clonal lines were generated by limiting dilution to generate a THP-1 TRIM34 TRIM5 double KO clonal cell line. KO efficiency was assessed by ICE analysis (ICE KO score = 72%, Additional file 1: Chromatograms S6–S10). Cells were doubly transduced with doxycycline-inducible, HA-tagged human TRIM34 or empty vector control in tandem with doxycycline-inducible, FLAG-tagged human TRIM5α, TRIM5Δv1, TRIM5 R332P, or empty vector control. Expression was induced in the presence of 125 ng/mL doxycycline, and expression levels were visualized by immunoblotting 72 h post-induction. b–d. THP-1 TRIM34 TRIM5 double KO clonal cells co-expressing doxycycline-inducible human TRIM34 or empty vector control and human TRIM5α (grey bars), TRIM5Δv1 (orange bars), TRIM5 R332P (blue bars), or empty vector control were infected with SIV_AGM-SAB (b), HIV-1 N74D (c), or HIV-1 (d) CA 1 day post-induction. Levels of infection were quantified 2 dpi by flow cytometry. Relative infectivity in induced cells (solid bars) is normalized to average infectivity in uninduced control cells (not shown). Fold inhibition is indicated where applicable. 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. One-sided p values were calculated by Welch’s t-test. ns nonsignificant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001