Project description:Although HIV-1 integration sites are considered to favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites have shown that the virus can integrate in a variety of host genomic locations, including non-genic regions, challenging the traditional understanding of HIV-1 integration site selection. Here, we showed that HIV-1 targets R-loops, a genomic structure made up of DNA–RNA hybrids, for integration. HIV-1 initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into regions of HIV-1-induced R-loops. Using a cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the presence of R-loops, regardless of transcriptional activity, directs HIV-1 integration targeting sites. We also found that HIV-1 integrase proteins bind to the host genomic R-loops. These findings provide fundamental insights into the mechanisms of retroviral integration and the new strategies of antiretroviral therapy against HIV-1 latent infection.
Project description:Although HIV-1 integration sites are considered to favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites have shown that the virus can integrate in a variety of host genomic locations, including non-genic regions, challenging the traditional understanding of HIV-1 integration site selection. Here, we showed that HIV-1 targets R-loops, a genomic structure made up of DNA–RNA hybrids, for integration. HIV-1 initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into regions of HIV-1-induced R-loops. Using a cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the presence of R-loops, regardless of transcriptional activity, directs HIV-1 integration targeting sites. We also found that HIV-1 integrase proteins bind to the host genomic R-loops. These findings provide fundamental insights into the mechanisms of retroviral integration and the new strategies of antiretroviral therapy against HIV-1 latent infection.
Project description:Although HIV-1 integration sites are considered to favor active transcription units in the human genome, high-resolution analysis of individual HIV-1 integration sites have shown that the virus can integrate in a variety of host genomic locations, including non-genic regions, challenging the traditional understanding of HIV-1 integration site selection. Here, we showed that HIV-1 targets R-loops, a genomic structure made up of DNA–RNA hybrids, for integration. HIV-1 initiates the formation of R-loops in both genic and non-genic regions of the host genome and preferentially integrates into regions of HIV-1-induced R-loops. Using a cell model that can independently control transcriptional activity and R-loop formation, we demonstrated that the presence of R-loops, regardless of transcriptional activity, directs HIV-1 integration targeting sites. We also found that HIV-1 integrase proteins bind to the host genomic R-loops. These findings provide fundamental insights into the mechanisms of retroviral integration and the new strategies of antiretroviral therapy against HIV-1 latent infection.
Project description:SON and SRRM2 were depleted in U2OS and HeLa cells and infected with HIV-1. Similarly HEK293T cells were infected with HIV-1 virus.
Project description:SON and SRRM2 were depleted in U2OS and HeLa cells and infected with HIV-1. Similarly HEK293T cells were infected with HIV-1 virus.
Project description:We used 4C sequencing technology for high-throughput profiling of HHV6A integration in both HEK293T cells and SMC cells. We identified genome-wide host-virus and virus-virus interactions profiles.
Project description:HIV-1 integration introduces ectopic transcription factor binding sites into host chromatin. We postulate that the integrated provirus serves as an ectopic enhancer that recruits additional transcriptional factors to the integration locus, increases chromatin accessibility, changes 3D chromatin interactions, and enhances both retroviral and host gene expression. We used 4 well-characterized HIV-1-infected cell line clones having unique integration sites and low to high levels of HIV-1 expression. Using single-cell DOGMA-seq, which captured the heterogeneity of HIV-1 expression and host chromatin accessibility, we found that HIV-1 transcription correlated with HIV-1 accessibility and host chromatin accessibility. HIV-1 integration increased local host chromatin accessibility within ~5–30 kb distance. CRISPRa and CRISPRi-mediated HIV-1 promoter activation and inhibition confirmed integration site-dependent HIV-1-driven changes of host chromatin accessibility. HIV-1 did not drive chromatin confirmation changes at the genomic level (by Hi-C) or the enhancer connectome (by H3K27Ac HiChIP). Using 4C-seq to interrogate HIV-1-chromatin interactions, we found that HIV-1 interacted with host chromatin ~100–300 kb from the integration site. By identifying chromatin regions having both increased transcription factor activity (by ATAC-seq) and HIV-1-chromtain interaction (by 4C-seq), we identified enrichment of ETS, RUNT, STAT, and ZNF transcription factor binding that may mediate HIV-1-host chromatin interactions. Our study found that HIV-1 promoter activity increased host chromatin accessibility, increased HIV-1-host chromatin interactions in an integration site dependent manner, within the existing chromatin boundaries without impacting broader host chromatin structure.
Project description:U1 monocytic cell line was cloned from U937 cells that survived an infection with Human Immunodeficiency Virus HIV-1 and bear integration sites, serving as one of the most studied HIV latent models. Under the hypothesis that the hyperdopaminergic environment of the brain of substance users affects HIV-infected cells and latency, we tested the effects of Dopamine and two dopamine receptors that were identified as able to signal changes ininnate immune cells, DRD1 and DRD4.
Project description:To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected human HEK293T, LKO (PSIP1-null), and LHKO (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to human genome.
Project description:The restructuring of chromatin architecture following lentiviral integration is not well elucidated. We jointly interrogate (HIV-distal & -local) chromatin organization (via Hi-C & ATAC-seq) and the RNA landscape around defined sites of proviral integration using HIV-inducible cellular models. We report chromatin interaction networks and nuclear ultrastructure around integrated HIV-1 are predominantly preserved, suggesting HIV integration does not induce large scale remodeling of cellular chromatin. Instead, we find that induction of proviral transcription leads to stark local changes in nucleosome organization with chromatin accessibility increasing at the intergenic junction between the HIV-1 3’ LTR and flanking cellular genome. This result suggests subtle changes in chromatin structure may be mediating proviral activation. Using long-read Nanopore RNA-seq, we interrogate the local host & HIV transcriptomes, observing a small fraction of HIV-1 transcripts are chimeric read-through products, where transcription initiates at the HIV-1 5’ LTR promoter and continues extensively into the flanking cellular genome. Despite provirus-driven read-through, HIV-1 appears to have only a modest effect on the local transcriptional environment. The changes in chromatin accessibility and read-through at activated proviruses closely resembles lytic Herpes simplex virus type 1 (HSV-1) induced cellular chromatin reprogramming. We propose chromatin “opening” at the 3’ LTR HIV-host junction is important for sustained proviral activity, and overall, HIV proviruses do not significantly alter local host transcription and chromatin structure. Our studies provide the first in-depth integrative investigation of 3D chromatin organization, nucleosome density, and HIV-host transcriptomes at HIV-host genic boundaries.