Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection

Lange, Ulrike; Białek, J; Walther, Thomas; Hauber, Joachim

doi:10.1016/j.virusres.2018.03.007

Cited by 5 publications

(4 citation statements)

References 22 publications

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“…This silencing was reversible as shown by the treatment of cell clones harbouring silenced HIV-1 promoters with TNF-α and Romidepsin that led to HIV-1 promoter reactivation. These observations are partly in line with Lange et al(32): They observed a comparable reversible silencing of the HIV-1 promoter in BACH2 within 20-40 days targeting two sites within BACH2 intron 5 via CRISPR/Cas9 and inserting one reporter gene under the control of the HIV-1 LTR-promoter in the same transcriptional orientation as BACH2 (Lange et al 2018). Here, we observed the same phenotypes in another intron of BACH2 and independent of the orientation of the HIV-1 integration.…”

Section: Discussionsupporting

confidence: 90%

Peer Review #1 of "HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells (v0.1)"

Bell¹

2020

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Background. The persistence of the latent HIV-1 reservoir is a major obstacle to cure 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 characterized. 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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Section: Discussionsupporting

confidence: 90%

Peer Review #1 of "HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells (v0.1)"

Bell¹

2020

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“…This silencing was reversible as shown by the treatment of cell clones harbouring silenced HIV-1 promoters with TNF-a and Romidepsin that led to HIV-1 promoter reactivation. These observations are partly in line with Lange et al (2018): They observed a comparable reversible silencing of the HIV-1 promoter in BACH2 within 20-40 days targeting two sites within BACH2 intron 5 via CRISPR/Cas9 and inserting one reporter gene under the control of the HIV-1 LTR-promoter in the same transcriptional orientation as BACH2. Here, we observed the same phenotypes in another intron of BACH2 and independent of the orientation of the HIV-1 integration.…”

Section: Discussionsupporting

confidence: 85%

HIV-1 promoter is gradually silenced when integrated intoBACH2in Jurkat T-cells

Inderbitzin

Kok

Jörimann

et al. 2020

PeerJ

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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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“…Proviruses are known to induce oncogenic modification of genes by the insertion of a promoter or an enhancer, which leads to the overexpression of a host oncogene and/or a truncation of the encoded protein [57]. Similar mechanisms likely affect the expression of the three genes (BACH2, MKL2,and STAT5B) previously identified as targets for HIV provirus mediated preferential expansion or survival of infected T cells in humans [21,23,28,36,58]. In the present study, we did a comprehensive search for additional genes in which an HIV provirus insertion could cause similar posititve selection for infected T cells.…”

Section: Plos Pathogensmentioning

confidence: 99%

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

et al. 2021

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HIV persists during antiretroviral therapy (ART) as integrated proviruses in cells descended from a small fraction of the CD4+ T cells infected prior to the initiation of ART. To better understand what controls HIV persistence and the distribution of integration sites (IS), we compared about 15,000 and 54,000 IS from individuals pre-ART and on ART, respectively, with approximately 395,000 IS from PBMC infected in vitro. The distribution of IS in vivo is quite similar to the distribution in PBMC, but modified by selection against proviruses in expressed genes, by selection for proviruses integrated into one of 7 specific genes, and by clonal expansion. Clones in which a provirus integrated in an oncogene contributed to cell survival comprised only a small fraction of the clones persisting in on ART. Mechanisms that do not involve the provirus, or its location in the host genome, are more important in determining which clones expand and persist.

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Pinpointing recurrent proviral integration sites in new models for latent HIV-1 infection

Cited by 5 publications

References 22 publications

Peer Review #1 of "HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells (v0.1)"

Peer Review #1 of "HIV-1 promoter is gradually silenced when integrated into BACH2 in Jurkat T-cells (v0.1)"

HIV-1 promoter is gradually silenced when integrated intoBACH2in Jurkat T-cells

Integration in oncogenes plays only a minor role in determining the in vivo distribution of HIV integration sites before or during suppressive antiretroviral therapy

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