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:Human immunodeficiency virus type-2 (HIV-2) establishes reservoirs at levels comparable to HIV-1, but its infection is associated with lower rates of progression to AIDS. As such, a hallmark of people living with HIV-2 is the very low—usually undetectable—viral loads, which is partially explained by a post-transcriptional repression exerted at the level of protein synthesis and the accumulation of the full-length RNA in cytoplasmic granules. Here, we investigated whether HIV-2 Gag expression was regulated by the presence of the m6A RNA modification. Our results indicate that the HIV-2 full-length RNA contains m6A residues, which are required for Gag synthesis. Interestingly, the YTHDF family of cytoplasmic m6A readers exert different effects on HIV-2 Gag synthesis. As such, while YTHDF1 and YTHDF2 are necessary for Gag synthesis, YTHDF3 negatively regulates Gag expression by promoting full-length RNA localization in cytoplasmic granules. Interestingly, we demonstrate that the cellular m6A machinery including writers, erasers and readers re-localize into cytoplasmic granules together with the full-length RNA in a non-Gag producing cell sub-population. We also observed that FTO overexpression has no effect in Gag synthesis but promotes full-length RNA packaging suggesting a dynamic regulation of the cytoplasmic fate of this viral RNA. Together, these data suggest that the HIV-2 full-length RNA undergoes a complex regulation exerted by the cellular m6A machinery that controls the cytoplasmic localization, ribosome association and particle incorporation of this viral RNA.
Project description:HIV-1 infection establishes a reservoir of long-lived cells with integrated proviral DNA that can persist despite antiretroviral therapy (ART). The mechanisms governing the transcriptional regulation of the provirus are complex and incompletely understood. Here, we investigated the role of histone H3 citrullination, a post-translational modification catalyzed by protein-arginine deiminase type-4 (PADI4), in HIV-1 transcription and latency. We found that PADI4 inhibition by GSK484 reduced HIV-1 transcription after T cell activation in ex vivo cultures of CD4 T cells from viremic and ART treated people living with HIV-1 (PLWH). The effect was more pronounced in the viremic group. Using cell models of HIV-1 latency, we showed that PADI4-mediated citrullination of histone H3 occurred at the HIV-1 promoter upon T cell stimulation which facilitated proviral transcription. HIV-1 preferentially integrated into genomic regions marked by H3 citrullination and these integrated proviruses were less prone to latency compared to those in non-citrullinated chromatin. Inhibiting PADI4 led to compaction of the HIV-1 promoter chromatin and an increase of HP1a-covered heterochromatin, in a mechanism partly dependent on the HUSH complex. Our data reveal a novel mechanism of HIV-1 transcriptional regulation by PADI4 through H3 citrullination.
Project description:Schlafen-5 (SLFN5) is an interferon-induced protein of the Schlafen family which are involved in immune responses and oncogenesis. To date, little is known regarding its anti-HIV-1 function. Here, the authors report that overexpression of SLFN5 inhibits HIV-1 replication and reduces viral mRNA levels, whereas depletion of endogenous SLFN5 promotes HIV-1 replication. Moreover, they show that SLFN5 markedly decreases the transcriptional activity of HIV-1 long terminal repeat (LTR) via binding to two sequences in the U5-R region, which consequently represses the recruitment of RNA polymerase Ⅱ to the transcription initiation site. Mutagenesis studies show the importance of nuclear localization and the N-terminal 1-570 amino acids fragment in the inhibition of HIV-1. Further mechanistic studies demonstrate that SLFN5 interacts with components of the PRC2 complex, G9a and Histone H3, thereby promoting H3K27me2 and H3K27me3 modification leading to silencing HIV-1 transcription. In concert with this, they find that SLFN5 blocks the activation of latent HIV-1. Altogether, their findings demonstrate that SLFN5 is a transcriptional repressor of HIV-1 through epigenetic modulation and a potential determinant of HIV-1 latency.