Excision of HIV-1 proviral DNA by recombinant cell permeable tre-recombinase

Abstract

Over the previous years, comprehensive studies on antiretroviral drugs resulted in the successful introduction of highly active antiretroviral therapy (HAART) into clinical practice for treatment of HIV/AIDS. However, there is still need for new therapeutic approaches, since HAART cannot eradicate HIV-1 from the infected organism and, unfortunately, can be associated with long-term toxicity and the development of drug resistance. In contrast, novel gene therapy strategies may have the potential to reverse the infection by eradicating HIV-1. For example, expression of long terminal repeat (LTR)-specific recombinase (Tre-recombinase) has been shown to result in chromosomal excision of proviral DNA and, in consequence, in the eradication of HIV-1 from infected cell cultures. However, the delivery of Tre-recombinase currently depends on the genetic manipulation of target cells, a process that is complicating such therapeutic approaches and, thus, might be undesirable in a clinical setting. In this report we demonstrate that E.coli expressed Tre-recombinases, tagged either with the protein transduction domain (PTD) from the HIV-1 Tat trans-activator or the translocation motif (TLM) of the Hepatitis B virus PreS2 protein, were able to translocate efficiently into cells and showed significant recombination activity on HIV-1 LTR sequences. Tre activity was observed using episomal and stable integrated reporter constructs in transfected HeLa cells. Furthermore, the TLM-tagged enzyme was able to excise the full-length proviral DNA from chromosomal integration sites of HIV-1-infected HeLa and CEM-SS cells. The presented data confirm Tre-recombinase activity on integrated HIV-1 and provide the basis for the non-genetic transient application of engineered recombinases, which may be a valuable component of future HIV eradication strategies.

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.