Biology subjects

Balfe, P.

Publications and source records attributed to Balfe, P..

8 recordsLinked to original sources

bioRxiv2026

XRN1-dependent RNA homeostasis regulates hepatitis D virus

Hepatitis delta virus (HDV) infection causes the most severe form of viral hepatitis, yet the host mechanisms restricting HDV replication remain poorly understood. Although HDV infection activates the MDA5-LGP2 pathway and induces robust interferon (IFN) responses, these antiviral programs have limited impact on viral replication. Here we identify the 5-3 exoribonuclease XRN1 as a key regulator of HDV infection. Loss of XRN1 in hepatoma cells increased double-stranded RNA (dsRNA) levels and induced interferon-stimulated genes (ISGs), including MDA5 and MAVS, and limited HDV infection. XRN1 deficiency enhanced ADAR1-mediated RNA editing, revealing a dynamic interplay between endogenous dsRNA metabolism and antiviral sensing. Inhibition of type I IFN signaling restored MDA5 expression and rescued HDV replication in XRN1-deficient cells. To identify the downstream effectors responsible for this restriction, we combined transcriptomic profiling with functional siRNA screening and identified DUSP5 and SIRPA as previously unrecognized ISGs with anti-HDV activity. Together, our findings establish XRN1 as a gatekeeper of dsRNA homeostasis that controls HDV replication through innate immune activation and reveals host antiviral effectors that may provide therapeutic opportunities for chronic hepatitis D.

molecular biology↗

bioRxiv2025

Hypoxia inducible factors regulate Pneumovirus replication by enhancing innate immune sensing

The immune mechanisms responsible for protection and pathogenesis in pneumoviral infection are not well defined. We demonstrated that pharmacological activation of the hypoxic inducible factor (HIF) signalling axis using Daprodustat limited viral replication through enhanced immune signalling. Transcriptomic analysis revealed HIF augmented activation of innate immune response genes, including interferon-stimulated gene 15 (Isg15), in the lung and spleen of mice infected with pneumonia virus of mice (PVM). In human respiratory syncytial virus (hRSV) infected airway epithelial cells, Daprodustat inhibited viral replication and enhanced ISG15 expression in a HIF-dependent manner. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. Moreover, Daprodustat increased interferon signalling in response to viral RNA, suggesting that HIF inhibits pneumovirus replication through enhancing viral RNA sensing. Mechanistically, Daprodustat reduced N6-methyladenosine modification of viral RNA through upregulation of RNA demethylases, promoting detection by innate immune sensors. This study highlights the intricate interplay between hypoxia and antiviral immunity and offers valuable insights into pneumovirus-host interactions and potential therapeutic interventions. SIGNIFICANCE STATEMENTThis study explores the role of the hypoxic inducible factor (HIF) signalling pathway in limiting pneumoviral infections, such as respiratory syncytial virus (hRSV) and pneumonia virus of mice (PVM). Using Daprodustat, a clinically approved activator of HIF signalling we demonstrated that stimulation of this pathway enhanced innate immune signalling and limited viral replication. Transcriptomic analysis showed that HIF promoted innate immune response genes in mice infected with PVM and human airway epithelial cells infected with hRSV. Importantly, inhibition of type I interferon signalling or the RIG-I sensing pathway abrogated the antiviral activity of HIF. We uncovered a role for HIF to regulate the levels of N6-methyladenosine modification of viral RNA transcripts, resulting in the increased activation of nucleic acid sensing.

microbiology↗

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

Circadian regulation of human immunodeficiency virus type 1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks curative therapies or vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organisms response to its environment and invading pathogens. Recent reports of diurnal variation in peripheral viral load in HIV-1 infected subjects highlights the need for mechanistic studies. Here, we demonstrate rhythmic HIV-1 replication in circadian-synchronised model systems and show the circadian transcription factors, BMAL1 and REV-ERB, bind conserved motifs in the HIV-1 promoter. REV-ERB competes with ROR for binding to the same regulatory motif, and we uncover a role for ROR inhibitors to perturb rhythmic HIV-1 replication and host gene expression. We demonstrate that many HIV-1 host factors are circadian regulated and likely to define rhythmic viral replication. In summary, this study increases our understanding of the mechanisms underlying the circadian regulation of HIV that can inform HIV therapy and management.

microbiology↗

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↗

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

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-hour oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components participating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, CLOCK and BMAL1 activate rhythmic transcription of genes including the nuclear receptor REV-ERB, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication, and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. Furthermore, the REV-ERB agonist SR9009 inhibited promoter activity of different HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic ligands to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock transcription factors in regulating HIV-1 replication.

microbiology↗