Biology subjects

Kirchherr, J. L.

Publications and source records attributed to Kirchherr, J. L..

3 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↗

bioRxiv2020

Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. ImportanceThe Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

microbiology↗