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


Inderbitzin, Anne; Kok, Yik Lim; Jörimann, Lisa; Kelley, Audrey; Neumann, Kathrin; Heinzer, Daniel; Cathomen, Toni; Metzner, Karin J (2020). HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells. PeerJ, 8:e10321.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Medical Virology
04 Faculty of Medicine > University Hospital Zurich > Clinic for Infectious Diseases
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Scopus Subject Areas:Life Sciences > General Neuroscience
Life Sciences > General Biochemistry, Genetics and Molecular Biology
Life Sciences > General Agricultural and Biological Sciences
Language:English
Date:2020
Deposited On:19 Jan 2021 16:01
Last Modified:09 Feb 2021 09:56
Publisher:PeerJ, Ltd.
ISSN:2167-8359
OA Status:Gold
Free access at:PubMed ID. An embargo period may apply.
Publisher DOI:https://doi.org/10.7717/peerj.10321
PubMed ID:33282555

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