A proof of principle study
Novel procedures to cure infections with human immunodeficiency virus (HIV-1) aim at eradicating ‘latently infected cells’. These cells carry the HIV genetic information (‘provirus’) integrated in their genomes but show a low or no viral gene expression. This characteristic makes these cells invisible for the immune system and to remain as ‘virus-reservoirs’. One eradication strategy is to induce (‘purge’) viral gene expression and to tag the cells for clearance by immune cells (‘shock and kill’). In our study, we applied a new approach using transcription activator-like effector proteins (TALEs) to specifically purge HIV gene expression. TALEs have a programmable DNA-specificity and accordingly were designed to target different regions of the HIV promoter. Indeed, some of these TALEs strongly activated transcription and viral outgrowth in HIV infected cultured cells as well as in cells of different infected patients. Accordingly, TALEs may be valuable tools in strategies aimed at removing HIV-1 reservoirs.
TALEs were originally characterized in plant-pathogenic bacteria that translocate these effectors into host cells to reprogram plant gene expression. The elucidation of their binding mode to DNA enabled the rapid and cost-effective generation of ‘designer’ TALEs, which are programmable to bind to a chosen DNA sequence and to activate transcription there. The idea for this study was triggered by the observation that TALEs induce transcription also in human cells making them a potentially interesting tool for ‘shock and kill’ procedures. The usage of TALEs was particularly attractive, because they should enable a specific (i.e. exclusive) activation of HIV gene expression. This is in contrast to ‘latency-reversing agents (LRAs)’ that are currently applied in clinical trials and that reveal considerable drawbacks as gene expression is globally induced.
Problems that occurred along the way to induce HIV gene expression by TALEs concerned a low expression efficiency of the proteins in HIV-infected T-cells and PBMCs; this was solved by using codon-optimized TALE genes and a lentiviral delivery system. Surprisingly, only a short region of the HIV promoter, the ‘HS2-region’ was accessible to TALE-mediated activation of HIV transcription (Figure 1). A major ‘aha!’ moment was the discovery that in comparison to the LRA Vorinostat (VOR), a clinically tested histone deacetylase inhibitor, the TALE induced HIV transcription and viral outgrowth were by far more efficient (Figure 1). Using customized engineered TALEs targeting HS2, this was shown in an HIV infected cell line as well as in primary cells obtained from different HIV-1 patients.
TALEs induce HIV-1 transcription. (a) HIV-1 ORFs and LTRs. The sequence of the HIV promoter region is shown; transcription factor binding sites and the promoter TATA box are underlined; the HS2 region is indicated. The target sequences of 13 TALEs are shown. (b, c) Intracellular levels of HIV-1 RNA in HIV infected T cells that expressed TALEs 1-13, TALE 14 (a non-specific TALE; negative control) and the green fluorescent protein (GFP; negative control). The TALEs were expressed via retroviruses and the RNA levels analyzed by qRT-PCR. The data are presented as fold change relative to the non-specific TALE 14 (negative control; HIV-1 RNA level set to 1). TALE-mediated transcription induction was compared to that obtained by Vorinostat (VOR) or DMSO (-) treatment. (d) Extracellular HIV-1 RNA levels in the supernatants of 8E5 that expressed TALE 5, TALE 14 or GFP or that were treated with VOR. qRT-PCR data are presented as fold change relative to the controls (TALE 14 or (-); HIV-1 RNA level set to 1). Error bars indicate standard deviations of three independent experiments, (**) P ≤ 0.01, (***) P ≤ 0.001.
About the authors
Pictured from left to right: Rene Geissler, Ilona Hauber, Jan Chemnitz, Joachim Hauber and Sven-Erik Behrens.
First author, Dr. Rene Geissler, studied Biochemistry in Halle/S in Germany and he did his Ph.D. work in the lab of Prof. Sven-Erik Behrens, where this publication was a part of. He now is a Post-Doctoral fellow at Cornell University (NY, USA).
Last author, Prof. Sven-Erik Behrens studied Biochemistry in Berlin, did his Ph.D. work on RNA splicing factors in the lab of Prof. Reinhard Lührmann in Marburg, Germany. He moved to Virology while being a Post-Doctoral fellow at the IRBM in Rome, Italy. Later, he ran his own labs as a PI at Giessen University, Germany and as an Associate Professor at Fox Chase Cancer Center in Philadelphia (PA, USA). He is a full Professor at the Martin Luther University in Halle/S since 2005.
About the research
Patient-adapted, specific activation of HIV-1 by customized TAL effectors (TALEs), a proof of principle study
Rene Geissler, Ilona Hauber, Nancy Funk, Annekatrin Richter, Martina Behrens, Ivonne Renner, Jan Chemnitz, Helga Hofmann-Sieber, Heidi Baum, Jan van Lunzen, Jens Boch, Joachim Hauber, Sven-Erik Behrens
Virology, Volume 486, December 2015, Pages 248–254