Abstract
BackgroundThe 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).MethodsWe 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.ResultsUpon 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].ConclusionSuccessful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity.
Highlights
Antiretroviral therapy (ART) efficiently blocks virus replication; it does not cure HIV-1 infection due to the presence of replication-competent but silenced proviruses preferentially integrated in long-lived resting CD4+ T-cells (Finzi et al, 1997; Wong et al, 1997)
This vector was inserted into introns 2 and 5 of BTB domain and CNC homology 2 (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
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
Summary
Antiretroviral therapy (ART) efficiently blocks virus replication; it does not cure HIV-1 infection due to the presence of replication-competent but silenced proviruses preferentially integrated in long-lived resting CD4+ T-cells (Finzi et al, 1997; Wong et al, 1997). One recurrent integration gene (RIG) that has been observed across patients in numerous independent studies is the gene BACH2, in which the provirus is almost exclusively found in intron 5 and in the same transcriptional orientation as BACH2 (Cesana et al, 2017; Ikeda et al, 2007; Imamichi et al, 2014; Mack et al, 2003; Maldarelli et al, 2014; Wagner et al, 2014) Since these BACH2 integration sites were identified in HIV-1-infected individuals who have been on ART for several years, it is conceivable that these proviruses are inactive, it remains unknown whether this presumed inactivity is a result of integration site-dependent silencing of replication-competent proviruses or due to defective proviruses. Conclusion: Successful targeted integration of the HIV-1-based vector LTatCL[M] allows longitudinal analyses of HIV-1 promoter activity
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