Figure 1.
Over expression of cell permeable Tre recombinases.
(A) Schematic representation of the Tre-recombinase constructs used in this study. (B) Coomassie stained SDS-PAGE (12%) of the E. coli expressed and purified proteins. (C) Western blot analysis of Tre-recombinase proteins using anti-Tre polyclonal antibodies. (D) Analysis of potential Tre-induced cellular toxicities. HeLa cells were exposed for 48 h to 1 µM of the indicated recombinant fusion proteins. Cellular metabolic activity was subsequently tested by alamarBlue assay. NC, negative control experiment in which Tre protein was omitted. Numbers over each bar indicates the p values calculated by paired t-test.
Figure 2.
Subcellular localization of cell permeable Tre in transduced HeLa cells.
Cellular uptake and localization of the indicated recombinant fusion proteins were studied by confocal laser scanning microscopy in HeLa cells. HeLa cells were exposed for 5 h to 1 µM of the various Tre-recombinases. Subsequently, the respective cell cultures were washed twice with PBS and PBS containing 0.5 mg per ml heparin for 5 min each. Nuclei were stained with Draq5 (blue label), Tre-recombinases (green label) with a primary polyclonal anti-Tre and secondary Cy2-labeled antibodies.
Figure 3.
Analysis of Tre activity in HeLa cells.
(A) Schematic diagram of the pSVLoxLTR reporter construct. The LoxLTR Tre target sequences and the P1 and P2 PCR primer sites, used for the monitoring of Tre-mediated recombination, are indicated. Recombination results in the de novo detection of a 724 bp PCR product. SV40 indicates the SV40 promoter. (B) HeLa cells were transiently transfected with pSVLoxLTR reporter plasmid, exposed to the indicated recombinant Tre proteins (lane 4–8) and recombination activity was monitored by PCR. M, DNA size markers; NC, negative control reaction lacking Tre; PC, positive control reaction in which Tre was coexpressed from the contransfected p3Tre plasmid. (C) Stable LoxLTR HeLa cells were treated with 1 µM of the indicated CPTR and recombination activity was analyzed as before.
Figure 4.
Protein stability of selected CPTR in mammalian cells.
Total cellular lysates were prepared at indicated time points from (A) HTLMNT and (B) HTatNT transduced and HIV-1 infected CEM-SS cells. Tre-recombinases were detected by Western analysis using anti-Tre polyclonal antibodies (upper panel). Equal sample loading was verified by detection of tubulin (lower panel). (C) Relative intensity of CPTR proteins at indicated time points.
Figure 5.
Interaction of CPTR with LoxLTR sites in living cells.
(A) Schematic representation of human genomic DNA containing HIV-1 provirus with flanking LTR. The Tre recombinase binding sequences, LoxLTR, shown in black box and red arrows indicates the primers used for PCR analysis. (B) ChIP assay of extracts derived from CPTR transduced and HIV-infected CEM-SS T cells. The Tre-specific LoxLTR target site was detected by PCR analysis. M, 1 kb NEB marker; T, 1∶10 diluted total input samples (positive PCR control); P, pull-down of Tre-recombinase using specific antibodies (α-Tre) or non-specific IgG. The LoxLTR-specific PCR products are shown.
Figure 6.
Excision of integrated HIV-1 proviral genomes.
(A) Depiction of the integrated proviral DNA and the products originating from Tre-mediated LoxLTR recombination. P1 and P2 denote PCR primer binding sites used for the detection of the excised circular recombination product. HIV-1 infected HeLa (B) and CEM-SS (C) cells were exposed to the indicated concentrations of recombinant HTLMNT protein. At 48 h post protein transduction genomic DNA was isolated and subjected to PCR. The recombination product is represented by the amplification of a 1.1 kB DNA fragment. NC, negative control in which HTLMNT was omitted; PC, positive control in which Tre was coexpressed from the p3Tre expression vector; M, DNA size markers.