Fig. 4.

RanBP2 regulates the anti-HIV-1 activity of rhesus TRIM5α. a HeLa cells were transduced with shBP2 or shE at d0, transduced with rhesus TRIM5α (T5) or empty vector (EV) at d1, and infected with VSV-G pseudotyped HIV-1 at MOI 1 at d5 in the absence or presence of the reverse transcription inhibitor Nevirapine (Nev). RanBP2 and TRIM5α transcripts were quantified by qRT-PCR and normalized for β2-microglobulin. To determine the efficiency of reverse transcription, Pol DNA transcripts were quantified at 6 hpi by qPCR and normalized for actin. Results show the mean of 3 experiments performed with independent lentiviral vector stocks ± SD. Unpaired t test was performed on HIV-1 data sets using Prism 6. Expression of rhTRIM5α led to a decrease in Pol copy numbers in control samples following HIV-1 infection, indicating efficient TRIM5α-mediated restriction. Depletion of RanBP2 cancelled the ability of TRIM5α to block HIV-1 reverse transcription, indicating that RanBP2 is key in mediating efficient TRIM5α restriction. b The same experiment was repeated but by first overexpressing rhTRIM5α then depleting RanBP2. TRIM5α expression was monitored by flow cytometry using HA labelling. Graphs are representative of two independent experiments. c To obtain a greater restriction phenotype, the experiment was repeated using rhTRIM5α cell clones. Graphs are representative of two independent experiments