HIV-1 transcription dominates over host gene activity at the HIV-1 integration site [ATAC-seq]

Despite effective antiretroviral therapy (ART), HIV-1 persists as an integrated DNA provirus in the genome of infected cells. Host cells can regulate HIV-1 transcription at the HIV-1 integration site dependent on the location (actively transcribed genes vs repressive chromatin) and the orientation (in the same vs opposite orientation of the host gene transcription) of HIV-1 integration. Presumably, HIV-1 follows the host gene transcriptional activity at the HIV-1 integration site. We interrogated HIV-1-host gene transcriptional interactions at the HIV-1 integration site using CRISPR-mediated activation and inhibition of the host genes (in which HIV-1 was integrated) in seven HIV-1-infected Jurkat T cell clones with known HIV-1 integration sites in the introns of actively transcribed genes and a non-genic region. Using ATAC-seq and strand-specific RNA-seq to examine chromatin accessibility and RNA transcription levels, we found that host gene activation did not increase HIV-1 transcription, while host gene inhibition did not decrease HIV-1 transcription. HIV-1 drove high levels of aberrant host RNA transcription regardless of the HIV-1 integration orientation. HIV-1-driven aberrant host RNA transcription was inhibited by CRISPR-mediated HIV-1 inhibition but not by CRISPR-mediated host gene activation or inhibition. When HIV-1 was integrated into a non-genic region, HIV-1 increased host chromatin accessibility and drove high levels of aberrant host RNA transcription. Overall, HIV-1 LTR promoter-driven transcriptional activity dominated over the host promoter activity. HIV-1 transcription does not passively follow host gene activity. Our results highlight that despite effective ART, silencing HIV-1 promoter is required to inhibit HIV-1-driven aberrant host gene expression and chronic immune activation. Overall design: Triplicate ATAC-seq profiling of Jurkat T cells infected with a single copy of replication-defective HIV-GFP, transduced with CRISPRa (dCas9-VP64 and MS2-P65-HSF1 transcription activator) or CRISPRi (dCas9-KRAB). Cell lines contain either nontargeting gRNA, HIV-targeting gRNA, or gRNA targeting the host gene containing the HIV integration site. Genomic DNA was prepared via ATAC-seq protocol and isolated by Qiagen MinElute PCR cleanup kit, (#28004) and library prep performed with NEBNext Ultra II Q5 master mix (NEB #M0544X) and Nextera indexed according to NEBNext Ultra II (NEB #E7645). Following baseline amplification, typically 8 cycles, SYBR qPCR was used to determine total cycle numbers needed. Typically, a total of 12 additional PCR cycles of library amplification were added, depending on qPCR results. After validating library size on an Agilent TapeStation, 150-bp paired-end sequencing was performed by NovaSeq 6000 at Yale Center for Genome Sequencing.