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

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.

Spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle as revealed by a genetic-insulators-containing dual-fluorescence HIV-1-based vector.

Long-lived latently HIV-1-infected cells represent a barrier to cure. We developed a dual-fluorescence HIV-1-based vector containing a pair of genetic insulators flanking a constitutive fluorescent reporter gene to study HIV-1 latency. The protective effects of these genetic insulators are demonstrated through long-term (up to 394 days) stable fluorescence profiles in transduced SUP-T1 cells. Analysis of 1,941 vector integration sites confirmed reproduction of HIV-1 integration patterns. We sorted monoclonal cells representing latent HIV-1 infections and found that both vector integration sites and integrity of the vector genomes influence the reactivation potentials of latent HIV-1 promoters. Interestingly, some latent monoclonal cells exhibited a small cell subpopulation with a spontaneously reactivated HIV-1 promoter. Higher expression levels of genes involved in cell cycle progression are observed in these cell subpopulations compared to their counterparts with HIV-1 promoters that remained latent. Consistently, larger fractions of spontaneously reactivated cells are in the S and G2 phases of the cell cycle. Furthermore, genistein and nocodazole treatments of these cell clones, which halted cells in the G2 phase, resulted in a 1.4-2.9-fold increase in spontaneous reactivation. Taken together, our HIV-1 latency model reveals that the spontaneous reactivation of latent HIV-1 promoters is linked to the cell cycle.