Fig. 2

TRIM22 inhibits Sp1-driven replication. a SupT1 cells were transduced with either pLKO.1/TRIM22_shRNA (TRIM22-KD) or pLKO.1/random_shRNA silencing control (CTRL-KD) lentiviral vectors and selected in culture by the addition of puromycin (0.2 µM). TRIM22 expression was assessed by absolute quantitative real-time PCR and normalized on the total number of 18S mRNA copies [4]. Specificity of TRIM22 knockdown was previously assessed [5]. Replication of the wild-type (b), tetO-CMV (c) and tetO-CMV-Sp1 (d) HIV-rtTA virus variants in TRIM22-KD and CTRL-KD SupT1 cell lines. Virus stocks were generated by transfection of 293T cells with DNA of the infectious clone. Cells were cultured in the presence of doxycycline (1 µg/mL) and virus-containing supernatant was harvested after 48 h and tested for Mg²⁺-dependent reverse transcriptase (RT) activity assay [4] yielding measurable amounts of RT activity (~4000 cpm/µL). Viral supernatants containing 1 × 10⁴ cpm-equivalents were added to 5 × 10⁵ SupT1 TRIM22-KD or KD-control cells and spinoculated at 2900 rpm for 2 h at 37 °C. Cells were cultured at 5 × 10⁵ cell/well in duplicate in the presence of doxycycline (1 µg/mL). Kinetics of viral replication were measured by RT activity assay in the supernatant collected every 3–4 days post-infection (PI) up to 32 days. Mean ± SEM of three independent infections in triplicates are shown