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. 2013;9(9):e1003587.
doi: 10.1371/journal.ppat.1003587. Epub 2013 Sep 26.

Highly Significant Antiviral Activity of HIV-1 LTR-specific Tre-Recombinase in Humanized Mice

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Free PMC article

Highly Significant Antiviral Activity of HIV-1 LTR-specific Tre-Recombinase in Humanized Mice

Ilona Hauber et al. PLoS Pathog. .
Free PMC article

Abstract

Stable integration of HIV proviral DNA into host cell chromosomes, a hallmark and essential feature of the retroviral life cycle, establishes the infection permanently. Current antiretroviral combination drug therapy cannot cure HIV infection. However, expressing an engineered HIV-1 long terminal repeat (LTR) site-specific recombinase (Tre), shown to excise integrated proviral DNA in vitro, may provide a novel and highly promising antiviral strategy. We report here the conditional expression of Tre-recombinase from an advanced lentiviral self-inactivation (SIN) vector in HIV-infected cells. We demonstrate faithful transgene expression, resulting in accurate provirus excision in the absence of cytopathic effects. Moreover, pronounced Tre-mediated antiviral effects are demonstrated in vivo, particularly in humanized Rag2⁻/⁻γc⁻/⁻ mice engrafted with either Tre-transduced primary CD4⁺ T cells, or Tre-transduced CD34⁺ hematopoietic stem and progenitor cells (HSC). Taken together, our data support the use of Tre-recombinase in novel therapy strategies aiming to provide a cure for HIV.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tre-mediated antiviral activity by conditional transgene expression.
(A) The HIV-derived lentiviral vector (LV) contains self-inactivating (SIN) long terminal repeats (LTR: ΔU3, R, U5), a Rev response element (RRE), central polypurine tract (cPPT), transgene cassette, post-regulatory element derived from woodchuck hepatitis virus (PRE), SV40 upstream polyadenylation enhancer elements (USE), splice donor (SD), splice acceptor (SA), and packaging (Ψ) signal, and the open reading frame for Tre-recombinase (tre) under the control of an HIV-LTR promoter containing two TAR elements (2TAR). In addition, the LV-Tre construct encodes the open reading frame for enhanced GFP (egfp). Expression of egfp is facilitated by the PGK promoter (PGK). (B) Schematic diagram of the replication–incompetent proviral reporter construct pNLT2ΔenvBLB. The open reading frame for the BFP-blasticidin-S deaminase fusion protein (blb) (blue box) partially substitutes the nef coding region. The 5′ and 3′ LTRs contain the Tre recombination site (loxLTR), a native HIV sequence. (C) Flow cytometric analysis of propidium iodide negative cells from one representative infection experiment follows the progression of GFP and BFP expression in polyclonal HeLa-smurf cells transduced with the indicated lentiviral constructs. (D) Tre and GFP expression was visualized by Western blot analysis. GAPDH served as a loading control.
Figure 2
Figure 2. Tre-mediated provirus excision and determination of Tre efficacy.
(A) Schematic depiction of Tre-mediated recombination. The PCR primers P1 and P2 amplify sequences from the provirus into the LTRs. Site-specific Tre-mediated recombination leaves a single LTR in the genome (“genomic scar”) and excises a circular recombination product containing the P1 and P2 primer binding regions. (B) Genomic DNA isolated from HeLa-smurf cells transduced with LV-Tre or LV-Ctr was analyzed by PCR using the P1 and P2 primers to detect the circular recombination product (1 kb). Negative PCR control, mock; lane M: DNA marker; p.t., post transduction. (C) To detect the genomic scar, genomic DNA prepared from Tre-treated (Tre +) or Ctr-treated cells (Tre −) 3 days after transduction were used as templates for PCR using HiLo PCR. Negative PCR control, mock; lane M: DNA marker. (D) The LTR region in the circular recombination product was subjected to DNA sequencing, revealing the presence of a single LTR flanked by nef-derived and gag leader sequences. Tre-treatment resulted in precise loxLTR recombination (boxed). (E) Genomic DNA containing either a single full length proviral genome or the residual LTR (“genomic scar”) is subjected to nrLAM PCR using LTR-specific primers (LTRIII). Subsequently, LAM PCR products consisting of LTR and genomic host DNA (LTR/int; 146 bp) or LTR and proviral coding sequence (LTR/blb; 150 bp) were quantified by qPCR. (F) Semi-quantitative PCR analysis of LAM PCR products from Tre-treated cells (Tre +), Ctr-treated control cells (Tre −) or negative PCR control (Ø). Lane M: DNA marker. (G) Quantitative PCR determination of LTR/int and LTR/blb LAM PCR products from Tre-treated cells (Tre +) or Ctr-treated (Tre −) control cells. Given are the means of three independent PCR reactions.
Figure 3
Figure 3. Analysis of potential Tre-related cytopathic effects.
Exponentially growing Jurkat T cells were transduced with LV-cCtr, or LV-cTre encoding constitutively expressed Tre-recombinase, or mock transduced. After enriching GFP+ cells by FACS (LV-cCtr or LV-cTre), cells were cultured for up to 15 weeks. Every week cells were harvested and analyzed. (A) Western blot analysis of the indicated proteins using rabbit polyclonal anti-Tre serum and mouse anti-α-Tubulin antibodies. Protein signals were visualized using an Odyssey Infrared Imaging System (LI-COR). (B) Metabolic activity measured by MTT assay; (C) Apoptosis assessed by Annexin V assay at week 15 of constitutive Tre expression; (D) Cell cycle progression monitored by DNA staining at week 15 of constitutive Tre expression. (E) Functionality of human primary transduced CD4+ T cells tested by flow cytometric analysis of CD154 and IFNγ expression after PMA/ionomycin stimulation. (F) Secretion pattern of Th1-, Th2- and Th17-specific cytokines in primary transduced CD4+ T cells after PMA/ionomycin stimulation as determined by multiplex ELISA. The pattern matches that of non-transduced cells (mock). (G) IFNγ- and IL4-specific Elispot analysis of human primary transduced CD4+ T cells after PMA/ionomycin stimulation. (H) CD34+ HSC were transduced with LV-Tre (Tat inducible promoter configuration) and 100 cells were seeded into cytokine-containing methyl-cellulose. Culture dishes were incubated for 14 days at adequate conditions, before counting colonies. (I) HSC differentiation assay as in panel F using an LV constitutively expressing Tre-recombinase (LV-cTre).
Figure 4
Figure 4. Analysis of potential Tre-related genotoxic effects.
(A) SKY (spectral karyotyping) analysis of metaphase spreads isolated from primary human CD4+ T cells overexpressing Tre for 21 days. An RGB display of the 24-color SKY hybridization of a representative normal metaphase is shown. (B) Array-CGH analysis of DNA isolated from primary human CD4+ T cells overexpressing Tre compared to mock-transfected cells. A representative chromosome (Chr17) is shown. Normal log2 ratios of color intensities (−4 to +4) for each probe populate the chart. A heterozygous deletion would be indicated by a green dot with the value <−1. A heterozygous duplication would be indicated by a red dot with a value >0.66.
Figure 5
Figure 5. Assay of potential Tre-related off-target effects.
(A) Nucleotide sequences of genomic sites and their locations in the human genome (in brackets). Sequences are aligned to the Tre recognition site loxLTR. Nucleotides that differ from loxLTR are shown in red. (B) Representation of the recombination assay in E. coli. and in HeLa cells, respectively. The evolution vector pEVO-Tre-target contains two directly repeated recombinase target sites (loxLTR) or the sequences GS1, GS2, GS3, GS4, GS5, GS6, and GS7. In E. coli, Tre is expressed from the PBAD promoter upon induction with L-arabinose. The vector also contains the regulatory gene araC, and a chloramphenicol resistance marker (Cmr). Recombination at the target sites leads to deletion of the 700 bp intervening region. Locations of the PCR primer binding sites (F, R) for detection of recombination are indicated. (C) Agarose gel showing the activity of Tre on loxLTR and the lack thereof for the seven genomic sites GS1 to GS7 (lanes 3–9). Upper panel: Recombination assayed in E. Coli. BsrG I/Xba I restriction digest results in a 4.9 kb fragment for non-recombined plasmid (two triangles) and a 4.2 kb fragment for recombined product (one triangle). Recombination tests on loxLTR served as negative and positive control (lanes 1 and 2). −, non-induced; +, induced with 1 mg/ml L-arabinose; M, DNA marker lane. Lower panel: Recombination assayed in HeLa cells. PCR using primers F and R that anneal to the vector DNA results in a 0.4 kb product when recombination occurs, while the non-recombined template results in a 1.1 kb PCR product. −, cotransfection with pIRESneo (i.e. no Tre expression); +, cotransfection with pIRESneo-Tre .
Figure 6
Figure 6. Suppression of plasma viremia in mice engrafted with Tre-modified human CD4+ T cells.
Rag2−/−γc−/− mice were infected with HIV-1 after adoptive transfer of LV-Tre (T1–T11) or LV-Ctr (negative control)-transduced (T12–T18) unselected CD4+ T cell pools. Plasma viral load (red lines) of individual mice was determined at the indicated time points after infection (p.i., post infection). Percent of human CD45+CD4+ cells in the peripheral blood of the individual animal is indicated (blue lines).
Figure 7
Figure 7. Analysis of the HIV-1 infected mice engrafted with LV-transduced (LV-Tre or LV-Ctr) unselected human CD4+ T cell pools.
(A) Plasma viral load of individual mice at the indicated time points after infection is shown. The fold difference of change in baseline levels of HIV-1 RNA copies (set to 100%) in the mean (horizontal bars; paired two-tailed t-test), and the total number of animals analyzed in each cohort are indicated. (B) Flow cytometric detection of human CD45+CD4+ cells. (C) Lentiviral vector-derived GFP expressing human CD4+ cells in the peripheral blood of the animals (indicated in percent). (D) Immunohistochemical analysis of spleen sections prepared from mice engrafted with either LV-Tre (left panels) or LV-Ctr (right panels) transduced T cells and stained for human CD3 and HIV p24 antigen. Scale bars indicate 100 µm.
Figure 8
Figure 8. Tre-mediated antiviral effects in Rag2−/−γc−/− mice engrafted with Tre-transduced human CD34+ HSC.
Animals engrafted with LV-Tre (HSC1-HSC10) or LV-Ctr (negative control)-transduced (HSC11–HSC18) unselected cord blood-derived CD34+ HSC pools were infected with HIV-1 and analyzed over time. Plasma viral load (red lines) of individual mice was determined at the indicated time points after infection (p.i., post infection). Percent of human CD45+CD4+ cells in the peripheral blood of the individual animal is indicated (blue lines).
Figure 9
Figure 9. Suppression of plasma viremia in the Rag2−/−γc−/− mice engrafted with Tre-modified (LV-Tre) CD34+ HSC.
(A) Plasma viremia was determined at the indicated time points after infection of LV-Tre or LV-Ctr (negative control) transduced animals. The fold difference of change in baseline levels of HIV-1 RNA copies (set to 100%) in the mean (horizontal bars; paired two-tailed t-test), and the total number of animals analyzed in each cohort are indicated. (B) Human CD45+CD4+ cells and, (C) Transgenic GFP expressing human CD4+ cells in the peripheral blood of the animals (indicated in percent) were detected by flow cytometric analysis. (D) Immunohistochemical analysis of lymph node sections prepared from mice engrafted with either LV-Tre (left panels) or LV-Ctr (right panels) transduced HSC and stained for human CD3 and HIV p24 antigen. Scale bars indicate 50 µm.

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Grant support

This work was financially supported by a grant of the Federal Ministry of Education and Research (GO-Bio FKZ 0315090). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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