Figure 1
SUMOylation regulates rhTRIM5α-mediated HIV-1 restriction. A. HeLa cells were transfected or not with rhTRIM5α and with control siRNA or siRNA targeting SUMO1, Ubc9 or PIAS1, 48 h before being challenged by increasing doses of HIV-1/GFP. The proportion of GFP-positive cells was measured by flow cytometry 48 h post-transduction. The graph shows means of duplicate values +/- SD, representative of three independent experiments. *p < 0.001 using the Huber/White/sandwich variance-covariance robust estimator (linear regression coefficients R2 are indicated). The expression levels of HA-rhTRIM5α were evaluated by western-blot (top right) using anti-HA antibodies (3 F10, Roche). The efficiency of RNA silencing was estimated by quantitative RT-PCR analysis and western-blot using anti-SUMO1 (Santa-Cruz Biotechnology sc-9060), anti-Ubc9 (Abgent, AM1261a) and anti-PIAS1 (Abcam, ab32219) antibodies (bottom right). B. Wild-type or rhTRIM5α-expressing HeLa cells were transfected with His-SUMO1 before transduction with increasing doses of HIV-1/GFP. The proportion of GFP-positive cells was measured by flow cytometry 48 h post-transduction. The graph shows means of duplicate values +/- SD, representative of three independent experiments. *p < 0.001 using the Huber/White/sandwich variance-covariance robust estimator. The expression levels of HA-rhTRIM5α were evaluated by western-blot (top right). Cell extracts from HeLa untransfected or transfected with SUMO1 were also analyzed by western-blot for SUMO1 expression (bottom right). C. hTRIM5α interacts with SUMO1 in the cytoplasm and nuclei of HeLa cells. Paraformaldehyde-fixed cells were incubated with primary rabbit anti-TRIM5α [13] and mouse anti-SUMO1 (clone 21C7, DSHB) antibodies for 1 h at 37°C. After washing slides were incubated with Duolink PLA probes (Olink Biosciences) for 1 h at 37°C. Ligation of the connector oligonucleotides, rolling-circle amplification and detection of the amplified DNA products were done with Duolink Detection Reagents Red according to the manufacturer’s instructions. Nuclei were labelled with Hoechst. Images were acquired using an LSM510 inverted microscope with a 63× objective. Scale bar indicates 5 μm.