Biology subjects

Balasubramaniam, M.

Publications and source records attributed to Balasubramaniam, M..

6 recordsLinked to original sources

bioRxiv2025

Zolpidem restores sleep and slows Alzheimer's progression in a mouse model

STRUCTURED ABSTRACTO_ST_ABSINTRODUCTIONC_ST_ABSDeficits in Non-Rapid Eye Movement (NREM) sleep facilitate Alzheimers disease (AD) progression. Enhancing GABAergic signaling can restore sleep. Unbiased computational analysis identified zolpidem as high-affinity GABA receptor modulator facilitating chloride transport that could slow AD. METHODSZolpidems effects on sleep and Alzheimers progression were evaluated in young APP/PS1 mice. Sleep was monitored with EEG/EMG telemetry. Widefield imaging with voltage-sensitive dyes was used to track sleep-dependent brain rhythms. Multiphoton microscopy allowed assessments of amyloid plaque load and basal neuronal calcium levels. Behavioral assays were used to measure memory and cognitive function. RESULTSZolpidem restored NREM sleep and rescued sleep-dependent brain rhythm, slow oscillation. Zolpidem administration reduced cortical amyloid plaque burden, mitigated neuronal calcium overload, and enhanced sleep-dependent memory consolidation without adverse effects on locomotion. DISCUSSIONZolpidem effectively slowed Alzheimers progression in young APP/PS1 mice. This supports zolpidems therapeutic promise as an intervention strategy at early stages of AD.

neuroscience↗

bioRxiv2025

CPSF6 Promotes HIV-1 Preintegration Complex Function

Cleavage and polyadenylation specificity factor 6 (CPSF6) is part of the cellular cleavage factor I mammalian (CFIm) complex that regulates mRNA processing and polyadenylation. CPSF6 also functions as a HIV-1 capsid (CA) binding host factor and promotes viral DNA integration targeting into gene dense regions of the host genome. However, the effects of CPSF6 on the activity of the HIV-1 preintegration complex (PIC) - the machinery that carries out viral DNA integration to establish infection - is unknown. To study CPSF6s role in HIV-1 PIC function, we extracted PICs from cells depleted of CPSF6 or expressing a CPSF6 mutant that cannot bind to CA. These PICs exhibited significantly lower integration activity when compared to the control PICs. Addition of recombinant CPSF6 restored the integration activity of PICs extracted from the mutant cells, suggesting a direct role of CPSF6 in PIC function. To solidify CPSF6s effect on PIC function, we inoculated CPSF6-depleted and CPSF6-mutant cells with HIV-1 particles and measured viral DNA integration into the host genome. A significant reduction in viral integration in these cells was detected and this defect was not a consequence of reduced reverse transcription or nuclear entry. Additionally, mutant viruses deficient in CA-CPSF6 binding showed no integration defect in CPSF6 mutant cells. Finally, sequencing analysis revealed that HIV-1 integration in the CPSF6 mutant cells was significantly redirected from the gene dense regions of the host genome. Collectively, these results suggest that CPSF6-CA interaction regulates PIC function both in vitro and in infected cells. IMPORTANCEHIV-1 infection is dependent on the interaction of the virus with host factors. However, the molecular details of virus-host factor interactions are not fully understood. For instance, HIV-1 capsid provides binding interfaces for several host factors. CPSF6 is one such capsid-binding host factor, whose cellular function is to regulate mRNA processing and polyadenylation. Initial work identified a truncated cytosolic form of CPSF6 that restricted HIV infection by blocking viral nuclear entry. However, it is now established that the full-length CPSF6 primarily promotes integration targeting into gene dense regions of the host genome. Here we report that CPSF6-CA interaction promotes the activity of HIV-1 preintegration complexes (PICs). We also observed that disruption of CPSF6-CA binding in target cells significantly reduced viral integration and directed integration targeting away from gene-dense regions. These findings demonstrate a critical role for the CPSF6-CA interaction in PIC function and integration targeting.

biochemistry↗

bioRxiv2024

Cyclophilin A Facilitates HIV-1 DNA Integration

Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA+/+) and CypA-depleted (CypA-/-) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypAs effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA+/+ cells but not in CypA-/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA+/+ cells but not in CypA-/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA-/- cells had lower activity in vitro compared to those from CypA+/+ cells. PICs from cells depleted for CypA and TRIM5 also had lower activity, suggesting that CypAs effect on PIC activity is independent of TRIM5. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA+/+ and CypA-/-cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. ImportanceHIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

microbiology↗

bioRxiv2024

Three Prime Repair Exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural and conformational properties.

HIV-1 integration into the human genome is dependent on 3-processing of the reverse transcribed viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3-processed DNA remained resistant. Here, we describe the mechanism by which the 3-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3-processed DNA was significantly lower than the unprocessed HIV-1 DNA by TREX1. The efficiency of degradation (kcat/KM) of the 3-processed DNA was also significantly lower than the unprocessed DNA. Furthermore, the binding affinity (Kd) of TREX1 was markedly lower to the 3-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3-processed DNA. These results pinpoint the biochemical mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3-processed HIV-1 DNA.

biochemistry↗

bioRxiv2022

HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

HIV-1 DNA integration into the host chromosomes is carried out by the preintegration complex (PIC). The PIC contains the viral DNA, virally encoded integrase enzyme and other critical viral/host factors. The PIC-associated viral DNA is preferentially integrated into gene bodies of actively transcribing genes. Here, we identify a biochemical mechanism underlying the preference of PIC-mediated viral DNA integration (PIC-VDI). Specifically, we observed that the PIC-VDI into human chromatin is preferred over the genomic DNA. Surprisingly, nucleosome core particles without any histone modifications were not preferred for PIC-VDI when compared to the analogous naked DNA. However, PIC-VDI was markedly enhanced with nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to HIV-1 DNA integration preference. Interestingly, we observed that nucleosomes with flanking linker DNA promoted PIC-VDI in the presence of LEDGF/p75. We also discovered that nucleosomes with linker DNA and H3K36me3 served as the optimal substrate for PIC-VDI. Mapping of the integration sites within these substrates identified preference of specific regions of the nucleosome core DNA for integration. Finally, we provide biochemical and genetic evidence that histone H1 protein, that condenses the chromatin, negatively regulates HIV-1 DNA integration, consistent with the integration preference for open chromatin structure. Collectively, these results identify the role of specific chromatin marks that drive HIV-1 integration preference and define the optimal substrate requirement for efficient DNA integration by the PIC.

microbiology↗

bioRxiv2021

HIV-1 Mutants that Escape the Cytotoxic T-Lymphocytes are Defective in Viral DNA Integration

ABSTRACT HIV-1 replication is durably controlled in certain untreated HIV-1-infected individuals expressing particular human leukocyte antigens (HLA). These HLAs tag infected cells for elimination by presenting specific viral epitopes to CD8+ cytotoxic T-lymphocytes (CTL). In individuals expressing HLA-B27, CTLs primarily target the capsid protein (CA)-derived KK10 epitope. Selection of CA mutation R264K helps HIV-1 escape the CTL response but severely diminishes virus infectivity. Here we report that the R264K mutation-associated infectivity defect arises primarily from impaired viral DNA integration. Strikingly, selection of the compensatory CA mutation S173A or depletion of host cyclophilin A largely rescues the R264K-associated integration and infectivity defects. Collectively, our study reveals novel mechanistic insights into the fitness defect incurred by an HIV-1 variant escaping a CA-directed CTL response.

microbiology↗