Zinc-Finger Nucleases Induced by HIV-1 Tat Excise HIV-1 from the Host Genome in Infected and Latently Infected Cells

Abstract

Highly active anti-retroviral therapy (HAART) cannot clear infected cells harboring HIV-1 proviral DNA from HIV-1-infected patients. We previously demonstrated that zinc-finger nucleases (ZFNs) can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells by targeting long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFN expression can be activated by the HIV-1 (Trans-Activator of Transcription) Tat protein. Our results show that functional expression of ZFNs induced by Tat excise the integrated proviral DNA of HIV-NL4-3-eGFP in approximately 30% of the population of HIV-1-infected cells. The results from HIV-1-infected human primary T cells and latently infected T cells treated with the inducible ZFNs further validated that proviral DNA can be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells.

Keywords: HIV-1; Tat; gene therapy; genome editing; genomic excision; inducible; zinc-finger nuclease.

Figure 1

Transient Luciferase Assay Detection of the Activity of the LTR or LTR-2 × TAR Promoter (A) Schematics of reporter gene luciferase expression cassettes. Expression of the luc gene driven by LTR or LTR-2 × TAR was very low in the absence of Tat protein but robustly increased in the presence of Tat protein. (B) Transient luciferase assay detection. HEK293T cells were co-transfected with the pLTR-luc or pLTR-2 × TAR-luc plasmid with the pcDNA3.1(−) or Tat expression plasmid pCMV-Tat and the internal control pRL-SV40 plasmid at the indicated time. The relative luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA) 72 hr post-transfection. The data were analyzed by normalizing the Tat-transfected group to the pcDNA3.1(−)-transfected group. Data represented the mean ± SD of three independent experiments.

Figure 2

Analysis of Different Doses of Tat-Induced Effects on Luciferase Gene Expression (A) Detection of LTR-driven luciferase gene expression when transfected with different doses of Tat. HEK293T cells were transfected with pLTR-luc in the presence of different doses of Tat at the indicated times. HEK293T cells transfected with pLTR-luc and pcDNA3.1(−) were used as controls. The relative luciferase activity was measured after 72 hr transfection. The data shown were normalized to the pcDNA3.1(−)-transfected group. Data represent the mean ± SD of three independent experiments. (B) Detection of LTR-2 × TAR-driven luc gene expression at different transfection amounts of Tat. Cell transfection methods were as indicated as above. Data represent the mean ± SD of three independent experiments. ***p < 0.001; paired t test.

Figure 3

Inducible ZFNs Could Induce HIV-1 Proviral DNA Excision by Transient Luciferase Assay HEK293T cells were co-transfected with pLTR-ZFN or pLTR-2 × TAR-ZFN with the HIV-1 gene expression construct carrying the luc gene pHIV-NL4-3-luc and pRL-SV40 at the indicated time. HEK293T cells transfected with ZFN expression plasmids pEF1a-ZFN, pHIV-NL4-3-luc, and pRL-SV40 were used as a positive control; HEK293T cells transfected with pcDNA3.1(−) or the ZFN empty expression plasmid pLTR or pLTR-2 × TAR, pHIV-NL4-3-luc, and pRL-SV40 were used as a negative control. The relative luciferase activity was measured after 72 hr transfection. The data shown were normalized to the pcDNA3.1(−)-transfected group. Data represent the mean ± SD of three independent experiments. ***p < 0.001; paired t test.

Figure 4

Inducible ZFNs Mediated HIV-1 Proviral DNA Excision in HIV-1-Infected Cells YA YA were left untransfected (mock) or nucleofected with pcDNA3.1(−), pLTR-ZFN, or pLTR-2 × TAR-ZFN at specified times. The percentage of EGFP⁺ cells was detected at different times post-transfection by flow cytometry. Data are representative of three independent experiments.

Figure 5

Inducible ZFNs Had No Effects on Cell Viability HIV-1-infected cells YA were transfected with pcDNA3.1(−), pLTR-ZFN, or pLTR-2 × TAR-ZFN at the indicated time. At different times post-transfection, YA viability was measured using the CCK-8 kit. The data show the cell viability of the pcDNA3.1(−)-treated group divided by that of the pLTR-ZFN or pLTR-2 × TAR-ZFN- transfected groups. The data represent the mean ± SD of three independent experiments.

Figure 6

Inducible ZFNs Mediated Excision of Integrated HIV-1 Proviral DNA in Human Primary T Cells HIV-1 NL4-3 infected CD4⁺ T Cells were nucleofected with 5 μg of pcDNA3.1(−), pLTR-ZFN, or pLTR-2 Χ TAR-ZFN at the indicated times. Culture supernatant was harvested for detecting HIV-1 p24 production after 72 hr transfection. Data are representative of three independent experiments, and error bars represent SD. ***p < 0.001; paired t test.

Figure 7

Inducible ZFNs Induced HIV-1 Proviral DNA Excision in HIV-1 Latently Infected Cell Clone C11 Cells (A) Schematic representation of regulated ZFN-mediated genome deletions. The light blue box in the 5′ and 3′ LTR of the HIV-1 genome (10.4 kb) indicates ZFN target sites. F-HG and R-HG (arrows), located outside of the integrated HIV-1 genome, were used for amplifying genome deletion events. (B) PCR products validated regulated ZFN-induced HIV-1 proviral genome deletion in the presence of Tat. C11 cells were nucleofected with pLTR-ZFN or pLTR-2 × TAR-ZFN with pcDNA3.1(−) or pCMV-Tat at the specified time. Genomic DNA was extracted 72 hr post-transfection and then subjected to PCR using the F-HG and R-HG primers.