Biology subjects

Turner, A.-M. W.

Publications and source records attributed to Turner, A.-M. W..

3 recordsLinked to original sources

bioRxiv2023

The histone methyltransferase SETD2 regulates HIV expression and latency through a post-transcriptional mechanism.

HIV can enter a state of transcriptional latency in CD4 T cells, allowing the virus to evade the host immune system and persist during antiretroviral therapy. Thus, understanding the mechanisms that drive HIV latency, and developing strategies to reactivate viral expression in latently infected cells, are key goals for achieving a cure for HIV. The full spectrum of mechanisms behind the regulation of HIV expression and latency are unclear but include covalent modifications to cellular histones that are associated with the integrated provirus. Here we investigate the role of the SETD2 histone methyltransferase, which deposits H3K36 trimethylation (H3K36me3) cotranscriptionally at genes, in HIV infection. We show that prevention of H3K36me3 through addition of a potent and selective inhibitor of SETD2 (EPZ-719) in human T cells leads to reduced post-integration viral gene expression and accelerated the emergence of a latently infected pool within a population of infected cells. CRISPR/Cas9-mediated knockout of SETD2 in HIV infected primary CD4 T cells confirmed the role of SETD2 in HIV expression. Intriguingly, EPZ-719 exposure also enhanced responsiveness of latently infected cells to latency reversal with the HDAC inhibitor vorinostat. Transcriptomic profiling of EPZ-719 exposed HIV-infected cells identified numerous cellular pathways impacted by EPZ-719. Finally, we show SETD2 inhibition does not affect HIV viral transcription, but instead leads to a shift in the pattern of viral RNA splicing - a result that would be predicted to reduce HIV expression. These results identify SETD2 and H3K36me3 as novel regulators of HIV expression and latency through a post-transcriptional mechanism.

microbiology↗

bioRxiv2023

A novel high throughput microwell outgrowth assay for HIV infected cells

Although antiretroviral therapy (ART) is highly effective at suppressing HIV replication, a viral reservoir persists that can reseed infection if ART is interrupted. Curing HIV will require elimination or functional containment of this reservoir, but the size of the HIV reservoir is highly variable between individuals. To evaluate the overall size of the HIV reservoir, several assays have been developed, including PCR based assays for viral DNA, the Intact Proviral DNA Assay (IPDA), and the Quantitative Viral Outgrowth Assay (QVOA). QVOA is the gold standard assay for measuring inducible replication competent proviruses, but this assay is technically challenging and time consuming. To provide a more rapid and less laborious tool for quantifying cells infected with replication competent HIV, we developed the Microwell Outgrowth Assay (MOA), in which HIV infected CD4 T cells are cocultured with an HIV-detecting reporter cell line in a polydimethylsiloxane (PDMS)/polystyrene array of nanoliter sized wells (rafts). Transmission of HIV from infected cells to the reporter cell line induces fluorescent reporter protein expression that is detected by automated scanning across the array. We show that this assay can detect HIV infected cells with a high degree of sensitivity and precision. Using this approach, we were able to detect HIV infected cells from ART-naive people with HIV (PWH) and from PWH on ART. Furthermore, we demonstrate that infected cells can be recovered from individual rafts and used to analyze the diversity of viral sequences. This assay may be a useful tool for quantifying and characterizing infected cells from PWH. Author summaryMeasuring the size of the HIV reservoir in people with HIV (PWH) will be important for determining the impact of HIV cure strategies. However, measuring this reservoir is challenging. We report a new method for quantifying HIV infected cells that involves culturing cells from PWH in an array of microwells with a cell line that detects HIV infection. We show that this approach can detect rare HIV infected cells and derive detailed virus sequence information for each infected cell.

microbiology↗

bioRxiv2021

Large-scale allosteric switch in the 7SK RNA regulates transcription in response to growth and stress

7SK is a highly conserved non-coding RNA that regulates eukaryotic transcription by sequestering positive transcription elongation factor b (P-TEFb). 7SK regulatory function likely entails changes in RNA structure, but characterizing dynamic RNA-protein complexes in cells has remained an unsolved challenge. We describe a new chemical probing strategy (DANCE-MaP) that uses maximum likelihood deconvolution and probabilistic read assignment to define simultaneously (i) per-nucleotide reactivity profiles, (ii) direct base pairing interactions, and (iii) tertiary and higher-order interactions for each conformation of multi-state RNA structural ensembles, all from a single experiment. We show that human 7SK RNA, despite significant heterogeneity, intrinsically codes for a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium is regulated by cell type, shifts dynamically in response to cell growth and stress, and can be exogenously targeted to modulate transcription in cells. Our data support that the 7SK structural ensemble functions as an integrator of diverse cellular signals to control transcription elongation in environment and cell specific ways, and establishes DANCE-MaP as a powerful strategy for comprehensively defining RNA structure and dynamics in cells.

biochemistry↗