Fig. 1.

Human and rhesus TRIM5α restrict LINE-1 retrotransposition. (A) HEK 293T cells were transfected with LINE-GFP or a retrotransposition-defective plasmid (JM111) and either empty vector or vector encoding human TRIM5α-HA, TRIM6-HA, TRIM22-HA, or TRIM34-HA. Five days posttransfection, GFP-positive cells were quantified by flow cytometry. Expression of the TRIM proteins was analyzed by immunoblot 2 d posttransfection. Membranes were probed with antibodies targeting the HA tag and the housekeeping gene HSP90. (B) HEK 293T cells were transfected with LINE-GFP together with the HA-tagged human TRIM5α, rhesus TRIM5 alleles Mamu1-5, TRIMCyp, or empty vector. Expression of the TRIM proteins was analyzed by immunoblot 2 d posttransfection. Membranes were probed with antibodies targeting the HA tag and the housekeeping gene HSP90. (C) HEK 293T cells were transfected with LINE-GFP and increasing amounts of human TRIM5α-HA or R437C-HA. Expression of the TRIM proteins was analyzed by immunoblot 2 d posttransfection. Membranes were probed with antibodies targeting the HA tag and the housekeeping gene HSP90. (D) HEK 293T cells expressing two different shRNAs targeting TRIM5α (shT5 #5 and shT5 #6) or scrambled shRNA (shC) were transfected with LINE-GFP. The knockdown of TRIM5α was confirmed by immunoblot using a TRIM5α-specific antibody. (E) ShC or shT5 #6 cells were transfected with LINE-GFP and WT TRIM5α or the shRNA-resistant variants TRIM5α^R-HA and R437C^R-HA. Expression of the TRIM5α proteins was confirmed by immunoblot using HA-specific antibodies. Five days posttransfection, GFP-positive cells were quantified by flow cytometry. The percentage of LINE-GFP-positive cells is shown as mean of triplicate transfections. Error bars represent SD. One out of three independent experiments is shown. (A, B, D, and E) Statistical analysis was done using one-way ANOVA followed by Tukey’s post hoc test or (C) two-way ANOVA followed by Bonferroni post hoc test. **P < 0.01; ***P < 0.001; ****P < 0.0001, n.s., not significant.