citations

                    Designed transcription activator-like effector proteins efficiently induced the expression of latent HIV-1 in latently infected cells
                

Authors:

Xiaohui Wang, Pengfei Wang, Zheng Fu, Haiyan Ji, Xiying Qu, Hanxian Zeng, Xiaoli Zhu, Junxiao Deng, Panpan Lu, Shijun Zha, Zhishuo Song, Huanzhang Zhu

In:

Source: Aids Research and Human Retroviruses
Publication Date: (2015)
Issue: 31(1): 98–106

Research Area:

Basic Research

Platform:

Nucleofector® I/II/2b

Experiment

Green fluorescent protein (GFP) expression visualization and flow cytometry analysis A total of 2×106 C11 cells or A10.6 cells were transfected with 2.0?µg of pcDNA3.1(-) or the TALE1-VP64 expression plasmid via nucleofection using the Amaxa Cell Line Nucleofector Kit V (Lonza, Gaithersburg, MD). The expression of GFP as a marker for the reactivation of the HIV-1 promoter was detected using a Nikon fluorescence microscope 24–72?h posttransfection. The images were captured using a Nikon E2 digital camera. At the indicated time points after transfection, the percentage of GFP-positive cells was measured via flow cytometry to determine the extent of reactivation. The cell suspension was centrifuged at 1000?rpm for 5?min, and after removal of the supernatant, the cell pellet was resuspended in 0.4?ml of phosphate-buffered saline (PBS). GFP expression was measured using a FACScan flow cytometer, and the FACS data were analyzed using CellQuest software (Macintosh, Sunnyvale, CA). A total of 10,000 gated events were collected, and the data represent the percentage of GFP-expressing cells out of the total gated events.

Abstract

HIV latency is the foremost barrier to clearing HIV infection from patients. Reactivation of latent HIV-1 represents a promising strategy to deplete these viral reservoirs. Here, we report a novel approach to reactivate latent HIV-1 provirus using artificially designed transcription activator-like effector (TALE) fusion proteins containing a DNA-binding domain specifically targeting the HIV-1 promoter and the herpes simplex virus-based transcriptional activator VP64 domain. We engineered four TALE genes (TALE1-4) encoding TALE proteins, each specifically targeting different 20-bp DNA sequences within the HIV-1 promoter, and we constructed four TALE-VP64 expression vectors corresponding to TALE1-4. We found that TALE1-VP64 effectively reactivated HIV-1 gene expression in latently infected C11 and A10.6 cells. We further confirmed that TALE1-VP64 reactivated latent HIV-1 via specific binding to the HIV-LTR promoter. Moreover, we also found that TALE1-VP64 did not affect cell proliferation or cell cycle distribution. Taken together, our data demonstrated that TALE1-VP64 can specifically and effectively reactivate latent HIV-1 transcription, suggesting that this strategy may provide a novel approach for anti-HIV-1 latency therapy in the future.

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