HIV-1 promoter is gradually silenced when integrated intoBACH2

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 geneBTB domain and CNC homology 2 (BACH2). We investigated HIV-1 promoter activity after integration into specific sites inBACH2. 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 ofBACH2of Jurkat T-cells via CRISPR/Cas9 technology in the same and convergent transcriptional orientation ofBACH2, and into the genomic safe harbour AAVS1. Single cell clones representing active (Cerulean⁺/mCherry⁺) and inactive (Cerulean⁻/mCherry⁺) HIV-1 promoters were characterized. Upon targeted integration of the 5.3 kb vector LTatCL[M] intoBACH2, active HIV-1 promoters were gradually silenced as reflected by decrease in Cerulean expression over a period of 162 days in culture. 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]. Our results show that the HIV-1 promoter is silenced when integrated intoBACH2without impairing BACH2 mRNA and protein expression. This might contribute to HIV-1 persistence, enabling infected T-cells to complete differentiation into a memory phenotype, persist, and clonally expand over time.