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Figure 1 | Virology Journal

Figure 1

From: Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection

Figure 1

Two-hybrid and co-precipitation assays show the requirement for hu -TOP1 residues E236/N237 for binding BTBD1 and BTBD2. A. The Proquest two hybrid system (Invitrogen) was used with clones as previously described [14]. The TOP1 cap domain [20] (aa 219-433) with either human residues E236/N237 (TOP1-EN) or AGM residues D236/S237 (TOP1-DS; three independent clones designated subscript 1, 2 and 3) fused to the Gal4 DNA binding domain (BD) were expressed from the minus Leu selectable plasmid. BTBD1 (aa 82-525) and BTBD2 (aa 1-482) were expressed as Gal4 activating domain (AD) fusions from the minus Trp selectable plasmid. Yeast transformed with these plasmids or with their parent plasmids without TOP1, BTBD1 or BTBD2 sequences (empty) were replica plated using three fold dilutions onto plates without Leu and Trp (-Leu,-Trp) to detect the presence of the plasmids. To detect the two hybrid protein interactions, the yeast were replica plated onto plates lacking Leu, Trp and His (and with 25 mM 3AT to adjust His selection stringency); plates lacking Leu, Trp and Ura (-Ura); or, plates lacking Leu and Trp and containing 5-fluoorotic acid (FOA, a substrate of URA3 that is converted into a toxic compound). hu-TOP1E236/N237 (TOP1-EN) shows interaction with BTBD1 and BTBD2 but three clones of hu-TOP1D236/S237 do not. B. GST-pulldown assay: GST fused to the cap domain of hu-TOP1 (EN, duplicate lanes; DS, triplicate clones) on glutathione beads were incubated with cell extracts containing C-terminal regions of BTBD1 (aa 186-482) and BTBD2 (aa 219-525). After incubation and washing, the bead pellets were processed for Western blots using polyclonal antibody recognizing both BTBD1 and BTBD2. GST-capTOP1E236/N237 co-precipitates BTBD1 (p-BTBD1) and BTBD2 (p-BTBD2) but GST-capTOP1D236/S237 does not. Immunostaining of GST-capTOP1 is with GST antibody (GST-TOP1-I/II). Lane 1 was loaded with 1/30th of the extract containing BTBD1 and BTBD2. C. As in "B" but endogenous BTBD1 and BTBD2 enriched from 293T cells is detected using peptide antibodies specific to each protein that were pulled down from extracts of 293T cells by GST-capTOP1E236/N237 but not by GST-capTOP1D236/S237. All experimental results were reproduced in at least two replicate experiments.

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