Biology subjects

Dash, C.

Publications and source records attributed to Dash, C..

8 recordsLinked to original sources

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

The MICOS Complex Regulates Mitochondrial Structure and Oxidative Stress During Age-Dependent Structural Deficits in the Kidney

Due to aging, the efficiency of kidney function begins to decrease. Dysfunction in mitochondria and their cristae is a hallmark of aging. Therefore, age-related decline in kidney function could be attributed to changes in mitochondrial ultrastructure, increased reactive oxygen species, and alterations in metabolism and lipid composition. We sought to understand how mitochondrial ultrastructure is altered over time in tubular kidney cells. A serial block facing-scanning electron microscope and manual segmentation using the Amira software were employed to visualize murine kidney samples during the aging process at 3 months (young) and 2 years (old). We found that 2-year mitochondria are more fragmented with many uniquely shaped mitochondria observed across aging, concomitant with shifts in ROS, metabolomics, and lipid homeostasis. Furthermore, we demonstrate that the mitochondrial contact site and cristae organizing system (MICOS) complex is impaired in the kidney during aging. Disruption of the MICOS complex resulted in altered mitochondrial metabolic function and increased ROS levels. We found significant, detrimental structural changes in the mitochondria of aged kidney tubules, suggesting a potential mechanism underlying the increased frequency of kidney disease with aging. We hypothesize that disruption of the MICOS complex exacerbates mitochondrial dysfunction, creating a vicious cycle of mitochondrial degradation and oxidative stress, which impacts kidney health. Impact and ImplicationsDue to aging, the efficiency of kidney function begins to decrease, and the risk of kidney diseases may increase; however, the specific regulators of mitochondrial age-related changes are poorly understood. This study demonstrates that the MICOS complex may be a target for mitigating age-related mitochondrial changes. The MICOS complex is associated with oxidative stress and calcium dysregulation, which also arise in many kidney pathologies. HighlightsO_LIAging alters the MICOS mRNA levels and disease markers. C_LIO_LIAging reduces cristae architecture, mitochondrial volume and complexity in murine kidney ultrastructure C_LIO_LIReducing MIC60 and CHCHD6 lowers Ca2+ uptake and retention and induces oxidative stress in HEK cells. C_LIO_LIMetabolomic Profiling revealed that NAD+ and amino acid metabolism were altered in aged kidneys. C_LIO_LIMICOS deficiency alters the reduced basal, ATP-linked, maximal capacity and spare capacity. C_LIO_LIDecreased modeled expression of CHCHD6 in individuals of European genetic ancestry is linked to chronic kidney disease, whereas decreased modeled expression of OPA1 in individuals of African genetic ancestry is associated with chronic kidney disease. C_LI Graphical AbstractKidney aging causes a decline in the MICOS complex, concomitant with metabolic, lipidomic, and mitochondrial structural alterations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/598108v3_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1a868f0org.highwire.dtl.DTLVardef@1817bfborg.highwire.dtl.DTLVardef@1f2a1a3org.highwire.dtl.DTLVardef@520692_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Monocytes Mobilized by Gut Neurons Remodel the Enteric Nervous System

The proper organization of the enteric nervous system (ENS) is critical for normal gastrointestinal (GI) physiology. Inflammatory bowel disease (IBD) dysregulates GI physiology, including bowel movements (motility), but in many IBD patients, GI motility disorders persist in remission through a poorly understood pathological process. Here we uncover that post-inflammatory GI dysmotility (PI-GID) stems from structural ENS remodeling driven by a combination of neuronal loss and neurogenesis. Enteric neurons respond to mucosal inflammation by upregulating CCL2 expression and facilitating the recruitment of CCR2+ monocytes into the neural myenteric plexus within the intestinal muscle. This is followed by the expansion of monocyte-derived macrophages and their migration into the myenteric ganglia and phagocytosis of neurons. However, excessive recruitment of monocytes results in disproportionate ENS remodeling and PI-GID. The expansion of inflammatory cells is known to promote tissue hypoxia. We find that enteric neurons become hypoxic upon colitis, but hypoxia-induced signaling via HIF1 initiates an adaptation program in enteric neurons to attenuate CCL2 expression and limit monocyte recruitment. We demonstrate that reinforcing HIF1 signaling in enteric neurons prevents PI-GID by reducing colitis-associated monocyte recruitment in the myenteric plexus and protecting against ENS remodeling. In summary, our findings unveil PI-GID pathogenesis and identify a regulatory axis for its prevention. One Sentence SummaryIntestinal mucosal inflammation engages enteric neurons in the inflammatory response leading to neurogenic recruitment of monocytes into the extra-mucosal myenteric plexus followed by pathological structural remodeling of the enteric nervous system by monocyte-derived macrophages.

immunology↗

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↗

bioRxiv2020

Phycobilins as potent food bioactive broad-spectrum inhibitor compounds against Mpro and PLpro of SARS-CoV-2 and other coronaviruses: A preliminary Study

In the twenty first century, we have witnessed three corona virus outbreaks; SARS in 2003, MERS in 2012 and ongoing pandemic COVID-19. To prevent outbreaks by novel mutant strains, we need broad-spectrum antiviral agents that are effective against wide array of coronaviruses. In this study, we scientifically investigated potent food bioactive broad-spectrum antiviral compounds by targeting Mpro and PLpro proteases of CoVs using in silico and in vitro approaches. The results revealed that phycocyanobilin (PCB) showed potential inhibitor activity against both proteases. PCB had best binding affinity to Mpro and PLpro with IC50 values of 71 m and 62 m, respectively. In addition, in silico studies of Mpro and PLpro enzymes of other human and animal CoVs indicated broad spectrum inhibitor activity of the PCB. Like PCB, other phycobilins such as phycourobilin (PUB), Phycoerythrobilin (PEB) and Phycoviolobilin (PVB) showed similar binding affinity to SARS-CoV-2 Mpro and PLpro

microbiology↗