Biology subjects

Archin, N. M.

Publications and source records attributed to Archin, N. M..

4 recordsLinked to original sources

bioRxiv2024

Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action.

microbiology↗

bioRxiv2024

BET Degraders Reveal BRD4 Disruption of 7SK and P-TEFb is Critical for Effective Reactivation of Latent HIV in CD4+ T-cells

HIV cure strategies that aim to induce viral reactivation for immune clearance leverage latency reversal agents to modulate host pathways which directly or indirectly facilitate viral reactivation. Inhibition of BET (bromo and extra-terminal domain) family member BRD4 reverses HIV latency, but enthusiasm for the use of BET inhibitors in HIV cure studies is tempered by concerns over inhibition of other BET family members and dose-limiting toxicities in oncology trials. Here we evaluated the potential for bivalent chemical degraders targeted to the BET family as alternative latency reversal agents. We observed that despite highly potent and selective BRD4 degradation in primary CD4+ T-cells from ART-suppressed donors, BRD4 degraders failed to induce latency reversal as compared to BET inhibitors. Further, BRD4 degraders failed to mimic previously observed synergistic HIV reactivation between BET inhibitors and an activator of the non-canonical NF-{kappa}B pathway. Mechanistic investigation of this discrepancy revealed that latency reversal by BET inhibitors is not related to the abatement of competition between Tat and BRD4 for P-TEFb, but rather the ability of BRD4 to disrupt 7SK and increase the levels of free P-TEFb. This activity is dependent on the shift of BRD4 from chromatin-bound to soluble and retargeting of P-TEFb to chromatin which is dependent on intact BRD4 but independent of the bromodomains.

molecular biology↗

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↗

bioRxiv2018

The SMAC Mimetic AZD5582 is a Potent HIV Latency Reversing Agent

The leading strategy towards eradication of human immunodeficiency virus (HIV) infection is the depletion of viral reservoirs through reversal of viral latency, followed by clearance of persistently infected cells. To date, a latency reversing agent (LRA) that reactivates a majority of the quiescent provirus population, without significant off-target effects, has not been identified. We show here that molecules mimicking the active N-terminal tetrapeptide of the second mitochondrial-derived activator of caspases (SMACm) potently reverse HIV latency in vitro and ex vivo without the pleotropic cellular effects seen with other LRA. We verified that SMACm facilitate latency reversal through activation of the non-canonical NFkB pathway as exemplified by rapid degradation of cellular inhibitor of apoptosis protein 1 (cIAP1), followed by a slower conversion of the inactive p100 form of NF{kappa}B2 into the active p52 transcription factor. A potent representative of this class, AZD5582, increases cell-associated HIV RNA expression in resting CD4+ T cells from ART-suppressed, HIV-infected donors while altering the expression of a restricted number of human genes. These findings represent the first demonstration that SMACm have single agent latency reversal activity in patient-derived cells and support evaluation of this class of molecules in preclinical animal models.

microbiology↗