Biology subjects

Wing, P. A.

Publications and source records attributed to Wing, P. A..

10 recordsLinked to original sources

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗

bioRxiv2024

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

bioRxiv2023

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. However, our knowledge of the host pathways that define susceptibility to RSV infection and disease severity is limited. Hypoxia inducible factors (HIFs) define cellular metabolic processes in response to low oxygen and are recognised to play a key role in regulating inflammatory responses in the lower respiratory tract. We demonstrate a role for hypoxia inducible factors (HIFs) to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors significantly reduce expression of the RSV entry receptor nucleolin and inhibit viral driven cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for treating this respiratory pathogen.

molecular biology↗

bioRxiv2023

The N6-methyladenosine demethylase ALKBH5 regulates the hypoxic HBV transcriptome.

Chronic hepatitis B is a global health problem and current treatments only suppress hepatitis B virus (HBV) infection, highlighting the need for new curative treatments. Oxygen levels influence HBV replication and we previously reported that hypoxia inducible factors (HIFs) activate the basal core promoter to transcribe pre-genomic RNA. Application of a probe-enriched long-read sequencing method to map the HBV transcriptome showed an increased abundance of all viral RNAs under low oxygen or hypoxic conditions. Importantly, the hypoxic-associated increase in HBV transcripts was dependent on N6-methyladenosine (m6A) modifications and an m6A DRACH motif in the 5 stem loop of pre-genomic RNA defined transcript half-life under hypoxic conditions. Given the essential role of m6A modifications in the viral transcriptome we assessed the oxygen-dependent expression of RNA demethylases and bio-informatic analysis of published single cell RNA-seq of murine liver showed an increased expression of the RNA demethylase ALKBH5 in the peri-central low oxygen region. In vitro studies with a human hepatocyte derived HepG2 cell line showed increased ALKBH5 gene expression under hypoxic conditions. Silencing the demethylase reduced the levels of HBV pre-genomic RNA and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIF expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen. Author SummaryOxygen levels influence HBV replication and hypoxia inducible factors (HIFs) activate HBV transcription. Long-read sequencing and mapping the HBV transcriptome showed an increased abundance of all viral RNAs under hypoxic conditions that was dependent on N6-methyladenosine modifications. Investigating the oxygen-dependent expression of RNA demethylases identified ALKBH5 as a hypoxic activated gene and silencing its expression showed a key role in regulating HBV and host gene expression under hypoxic conditions.

molecular biology↗

bioRxiv2022

An ACAT inhibitor regulates SARS-CoV-2 replication and antiviral T cell activity

The severity of disease following infection with SARS-CoV-2 is determined by viral replication kinetics and host immunity, with early T cell responses and/or suppression of viraemia driving a favourable outcome. Recent studies have uncovered a role for cholesterol metabolism in the SARS-CoV-2 life cycle and in T cell function. Here we show that blockade of the enzyme Acyl-CoA:cholesterol acyltransferase (ACAT) with Avasimibe inhibits SARS-CoV-2 entry and fusion independent of transmembrane protease serine 2 expression in multiple cell types. We also demonstrate a role for ACAT in regulating SARS-CoV-2 RNA replication in primary bronchial epithelial cells. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled in the acute phase of infection. Thus, re-purposing of available ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects.

immunology↗

bioRxiv2022

Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model.

Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.

microbiology↗

bioRxiv2021

Absolute quantitation of individual SARS-CoV-2 RNA molecules: a new paradigm for infection dynamics and variant differences

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/450133v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@10f7bf1org.highwire.dtl.DTLVardef@192214dorg.highwire.dtl.DTLVardef@c84916org.highwire.dtl.DTLVardef@1366287_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain. Highlights O_LISingle molecule quantification of SARS-CoV-2 replication uncovers early infection kinetics C_LIO_LIThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replication C_LIO_LIGenomic RNA is stable and persistent during the initial stages of infection C_LIO_LIB.1.1.7 variant replicates more slowly than the Victoria strain C_LI

microbiology↗

bioRxiv2021

Circadian regulation of SARS-CoV-2 infection in lung epithelial cells

The COVID-19 pandemic, caused by SARS-CoV-2 coronavirus, is a global health issue with unprecedented challenges for public health. SARS-CoV-2 primarily infects cells of the respiratory tract, via Spike glycoprotein binding angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organisms response to its environment and can regulate host susceptibility to virus infection. We demonstrate a circadian regulation of ACE2 in lung epithelial cells and show that silencing BMAL1 or treatment with a synthetic REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry. Treating infected cells with SR9009 limits viral replication and secretion of infectious particles, showing that post-entry steps in the viral life cycle are influenced by the circadian system. Transcriptome analysis revealed that Bmal1 silencing induced a wide spectrum of interferon stimulated genes in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to dampen SARS-CoV-2 infection. Our study suggests new approaches to understand and improve therapeutic targeting of SARS-CoV-2.

microbiology↗

bioRxiv2020

The CCCTC-binding factor CTCF represses hepatitis B virus Enhancer I and regulates viral transcription

Hepatitis B virus (HBV) infection is of global importance with over 2 billion people exposed to the virus during their lifetime and at risk of progressive liver disease, cirrhosis and hepatocellular carcinoma. HBV is a member of the hepadnaviridae family that replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate viral transcription. The chromatin-organising transcriptional insulator protein CCCTC-binding factor (CTCF) has been reported to regulate transcription in a diverse range of viruses. We identified two conserved CTCF binding sites in the HBV genome within Enhancer I and chromatin immunoprecipitation (ChIP) analysis demonstrated an enrichment of CTCF binding to integrated or episomal copies of the viral genome. siRNA knockdown of CTCF results in a significant increase in pre-genomic RNA levels in de novo infected HepG2 cells and those supporting episomal HBV DNA replication. Furthermore, mutation of these sites in HBV DNA minicircles abrogated CTCF binding and increased pre-genomic RNA levels, providing evidence of a direct role for CTCF in repressing HBV transcription. IMPORTANCEHepatitis B virus (HBV) is a global cause of liver disease. At least 300 million individuals are chronically infected with HBV, frequently leading to life-threatening liver cirrhosis and cancer. Following viral entry, HBV DNA enters the nucleus and is bound by histones that are subject to epigenetic modification. The HBV genome contains two enhancer elements that stimulate viral transcription but the interplay between the viral enhancers and promoters is not fully understood. We have identified the host cell protein CCCTC binding factor (CTCF) as a repressor of HBV gene expression. CTCF binds to the HBV genome within Enhancer I and represses transcription of pre-genomic RNA. These findings provide new insights into how HBV transcription is regulated and show a new role for CTCF as a transcriptional insulator by associating with the viral genome between Enhancer I and the downstream basal core promoter.

microbiology↗

bioRxiv2020

Hypoxic gene expression in chronic hepatitis B infected patients is not observed in state-of-art in vitro and mouse infection models

Hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC) worldwide. The prolyl hydroxylase domain (PHD)-hypoxia inducible factor (HIF) pathway is a key mammalian oxygen sensing pathway and is frequently perturbed by pathological states including infection and inflammation. We discovered a significant upregulation of hypoxia regulated gene transcripts in patients with chronic hepatitis B (CHB) in the absence of liver cirrhosis. We used state-of-the-art in vitro and in vivo HBV infection models to evaluate a role for HBV infection and the viral regulatory protein HBx to drive HIF-signalling. HBx had no significant impact on HIF expression or associated transcriptional activity under normoxic or hypoxic conditions. Furthermore, we found no evidence of hypoxia gene expression in HBV de novo infection, HBV infected human liver chimeric mice or transgenic mice with integrated HBV genome. Collectively, our data show clear evidence of hypoxia gene induction in CHB that is not recapitulated in existing models for acute HBV infection, suggesting a role for inflammatory mediators in promoting hypoxia gene expression.

microbiology↗