Figure 5. TRIM25 is required for KHNYN to inhibit HIV-1 with clustered CpG dinucleotides.
(A–B) HeLa Control CRISPR cells or TRIM25-G1 CRISPR cells were transfected with 500 ng pHIV-1 or pHIV-1EnvCpG86-561 and 500 ng of pGFP-FLAG or 62.5 ng pKHNYN-1-FLAG or pKHNYN-2-FLAG plus 437.5 ng of pGFP-FLAG. Culture supernatants from the cells were used to infect TZM-bl reporter cells (A). The bar charts show the average value of three independent experiments normalized to the value obtained for HeLa Control CRISPR cells co-transfected with pHIV-1 and pGFP-FLAG. Data are represented as mean ± SD. *p<0.05 as determined by a two-tailed unpaired t-test. p-values for GFP verses KHNYN-1 and KHNYN-2 for HIV-1EnvCpG86-561 in Control cells are 8.95 × 10−4 and 5.42 × 10−4, respectively. p-values for GFP verses KHNYN-1 and KHNYN-2 for HIV-1EnvCpG86-561 in TRIM25-G1 CRISPR cells are 1.78 × 10−3 and 1.01 × 10−4, respectively. Gag expression in the media as well as Gag, Hsp90, Env, Actin, KHNYN-FLAG and GFP-FLAG expression in the cell lysates was detected using quantitative immunoblotting (B). (C) Lysates of Control and TRIM25 CRISPR HEK293T cells transfected with pGFP-FLAG, pKHNYN-1-FLAG or pKHNYN-2-FLAG were immunoprecipitated with an anti-FLAG antibody. Post-nuclear supernatants and immunoprecipitation samples were analyzed by immunoblotting for HSP90, KHNYN-FLAG, TRIM25 and ZAP. The blots are representative of two independent experiments. (D) Lysates of Control and ZAP CRISPR HEK293T cells transfected with pGFP-FLAG, pKHNYN-1-FLAG or pKHNYN-2-FLAG were immunoprecipitated with an anti-FLAG antibody. Post-nuclear supernatants and immunoprecipitation samples were analyzed by immunoblotting for HSP90, KHNYN-FLAG, TRIM25 and ZAP. * indicates a non-specific band. The blots are representative of two independent experiments.