HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells.

Autor:	Inderbitzin A; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.; Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland., Kok YL; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland., Jörimann L; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.; Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland., Kelley A; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.; Life Science Zurich Graduate School, University of Zurich, Zurich, Switzerland., Neumann K; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland., Heinzer D; Institute for Neuropathology, University Hospital Zurich, Zurich, Switzerland.; Neuroscience Center Zurich Graduate School, University of Zurich, Zurich, Switzerland., Cathomen T; Institute for Transfusion Medicine and Gene Therapy, Medical Center, University of Freiburg, Freiburg, Germany.; Faculty of Medicine, University of Freiburg, Freiburg, Germany., Metzner KJ; Department of Infectious Diseases and Hospital Epidemiology, Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland.; Institute of Medical Virology, University of Zurich, Zurich, Switzerland.
Jazyk:	angličtina
Zdroj:	PeerJ [PeerJ] 2020 Nov 24; Vol. 8, pp. e10321. Date of Electronic Publication: 2020 Nov 24 (Print Publication: 2020).
DOI:	10.7717/peerj.10321
Abstrakt:	Background: The persistence of the latent HIV-1 reservoir is a major obstacle to curing HIV-1 infection. HIV-1 integrates into the cellular genome and some targeted genomic loci are frequently detected in clonally expanded latently HIV-1 infected cells, for instance, the gene BTB domain and CNC homology 2 (BACH2) . Methods: We investigated HIV-1 promoter activity after integration into specific sites in BACH2 in Jurkat T-cells. The HIV-1-based vector LTatCL[M] contains two fluorophores: (1) Cerulean, which reports the activity of the HIV-1 promoter and (2) mCherry driven by a constitutive promotor and flanked by genetic insulators. This vector was inserted into introns 2 and 5 of BACH2 of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation of BACH2 , and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean + /mCherry + ) and inactive (Cerulean - /mCherry + ) HIV-1 promoters were characterised. Results: Upon targeted integration of the 5.3 kb vector LTatCL[M] into BACH2 , the HIV-1 promoter was gradually silenced as reflected by the decrease in Cerulean expression over a period of 162 days. Silenced HIV-1 promoters could be reactivated by TNF-α and Romidepsin. This observation was independent of the targeted intron and the transcriptional orientation. BACH2 mRNA and protein expression was not impaired by mono-allelic integration of LTatCL[M]. Conclusion: Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity. Competing Interests: Karin J. Metzner has received travel grants and honoraria from Gilead Sciences, Roche Diagnostics, Tibotec, Bristol-Myers Squibb, and Abbott; the University of Zurich has received research grants from Gilead, Roche and Merck Sharp & Dohme for studies that Karin J. Metzner serves as principal investigator, and advisory board honoraria from Gilead Sciences. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All other authors declare no competing interests relevant to this study. (© 2020 Inderbitzin et al.)
Databáze:	MEDLINE
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