ASPP2 binds to hepatitis C virus NS5A protein via an SH3 domain/PxxP motif-mediated interaction and potentiates infection.

Hepatitis C virus (HCV) infects millions of people worldwide and is a leading cause of liver disease. Despite recent advances in antiviral therapies, viral resistance can limit drug efficacy and understanding the mechanisms that confer viral escape is important. We employ an unbiased interactome analysis to discover host binding partners of the HCV non-structural protein 5A (NS5A), a key player in viral replication and assembly. We identify ASPP2, apoptosis-stimulating protein of p53, as a new host co-factor that binds NS5A via its SH3 domain. Importantly, silencing ASPP2 reduces viral replication and spread. Our study uncovers a previously unknown role for ASPP2 to potentiate HCV RNA replication.

Hypoxia dampens innate immune signalling at early time points and increases Zika virus RNA levels in iPSC-derived macrophages.

Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIG-I)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under laboratory conditions of 18% oxygen (O2), that do not reflect the oxygen levels in most organs (1-5 % O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) infection. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Hypoxic conditions increased the abundance of ZIKV RNA highlighting the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defences.

The HCV Envelope Glycoprotein Down-Modulates NF-κB Signalling and Associates With Stimulation of the Host Endoplasmic Reticulum Stress Pathway.

Following acute HCV infection, the virus establishes a chronic disease in the majority of patients whilst few individuals clear the infection spontaneously. The precise mechanisms that determine chronic HCV infection or spontaneous clearance are not completely understood but are proposed to be driven by host and viral genetic factors as well as HCV encoded immunomodulatory proteins. Using the HIV-1 LTR as a tool to measure NF-κB activity, we identified that the HCV E1E2 glycoproteins and more so the E2 protein down-modulates HIV-1 LTR activation in 293T, TZM-bl and the more physiologically relevant Huh7 liver derived cell line. We demonstrate this effect is specifically mediated through inhibiting NF-κB binding to the LTR and show that this effect was conserved for all HCV genotypes tested. Transcriptomic analysis of 293T cells expressing the HCV glycoproteins identified E1E2 mediated stimulation of the endoplasmic reticulum (ER) stress response pathway and upregulation of stress response genes such as ATF3. Through shRNA mediated inhibition of ATF3, one of the components, we observed that E1E2 mediated inhibitory effects on HIV-1 LTR activity was alleviated. Our in vitro studies demonstrate that HCV Env glycoprotein activates host ER Stress Pathways known to inhibit NF-κB activity. This has potential implications for understanding HCV induced immune activation as well as oncogenesis.

Hypoxia-Inducible Factor 1 Alpha-Mediated RelB/APOBEC3B Down-regulation Allows Hepatitis B Virus Persistence.

BACKGROUND AND AIMS: Therapeutic strategies against HBV focus, among others, on the activation of the immune system to enable the infected host to eliminate HBV. Hypoxia-inducible factor 1 alpha (HIF1α) stabilization has been associated with impaired immune responses. HBV pathogenesis triggers chronic hepatitis-related scaring, leading inter alia to modulation of liver oxygenation and transient immune activation, both factors playing a role in HIF1α stabilization. APPROACH AND RESULTS: We addressed whether HIF1α interferes with immune-mediated induction of the cytidine deaminase, apolipoprotein B mRNA editing enzyme catalytic subunit 3B (APOBEC3B; A3B), and subsequent covalently closed circular DNA (cccDNA) decay. Liver biopsies of chronic HBV (CHB) patients were analyzed by immunohistochemistry and in situ hybridization. The effect of HIF1α induction/stabilization on differentiated HepaRG or mice ± HBV ± LTßR-agonist (BS1) was assessed in vitro and in vivo. Induction of A3B and subsequent effects were analyzed by RT-qPCR, immunoblotting, chromatin immunoprecipitation, immunocytochemistry, and mass spectrometry. Analyzing CHB highlighted that areas with high HIF1α levels and low A3B expression correlated with high HBcAg, potentially representing a reservoir for HBV survival in immune-active patients. In vitro, HIF1α stabilization strongly impaired A3B expression and anti-HBV effect. Interestingly, HIF1α knockdown was sufficient to rescue the inhibition of A3B up-regulation and -mediated antiviral effects, whereas HIF2α knockdown had no effect. HIF1α stabilization decreased the level of v-rel reticuloendotheliosis viral oncogene homolog B protein, but not its mRNA, which was confirmed in vivo. Noteworthy, this function of HIF1α was independent of its partner, aryl hydrocarbon receptor nuclear translocator. CONCLUSIONS: In conclusion, inhibiting HIF1α expression or stabilization represents an anti-HBV strategy in the context of immune-mediated A3B induction. High HIF1α, mediated by hypoxia or inflammation, offers a reservoir for HBV survival in vivo and should be considered as a restricting factor in the development of immune therapies.

Targeting human Acyl-CoA:cholesterol acyltransferase as a dual viral and T cell metabolic checkpoint.

Determining divergent metabolic requirements of T cells, and the viruses and tumours they fail to combat, could provide new therapeutic checkpoints. Inhibition of acyl-CoA:cholesterol acyltransferase (ACAT) has direct anti-carcinogenic activity. Here, we show that ACAT inhibition has antiviral activity against hepatitis B (HBV), as well as boosting protective anti-HBV and anti-hepatocellular carcinoma (HCC) T cells. ACAT inhibition reduces CD8+ T cell neutral lipid droplets and promotes lipid microdomains, enhancing TCR signalling and TCR-independent bioenergetics. Dysfunctional HBV- and HCC-specific T cells are rescued by ACAT inhibitors directly ex vivo from human liver and tumour tissue respectively, including tissue-resident responses. ACAT inhibition enhances in vitro responsiveness of HBV-specific CD8+ T cells to PD-1 blockade and increases the functional avidity of TCR-gene-modified T cells. Finally, ACAT regulates HBV particle genesis in vitro, with inhibitors reducing both virions and subviral particles. Thus, ACAT inhibition provides a paradigm of a metabolic checkpoint able to constrain tumours and viruses but rescue exhausted T cells, rendering it an attractive therapeutic target for the functional cure of HBV and HBV-related HCC.

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.

Circadian control of hepatitis B virus replication.

Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.

Estimating hepatitis B virus cccDNA persistence in chronic infection.

Hepatitis B virus (HBV) infection is a major global health problem with over 240 million infected individuals at risk of developing progressive liver disease and hepatocellular carcinoma. HBV is an enveloped DNA virus that establishes its genome as an episomal, covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Currently, available standard-of-care treatments for chronic hepatitis B (CHB) include nucleos(t)ide analogues (NAs) that suppress HBV replication but do not target the cccDNA and hence rarely cure infection. There is considerable interest in determining the lifespan of cccDNA molecules to design and evaluate new curative treatments. We took a novel approach to this problem by developing a new mathematical framework to model changes in evolutionary rates during infection which, combined with previously determined within-host evolutionary rates of HBV, we used to determine the lifespan of cccDNA. We estimate that during HBe-antigen positive (HBeAgPOS) infection the cccDNA lifespan is 61 (36-236) days, whereas during the HBeAgNEG phase of infection it is only 26 (16-81) days. We found that cccDNA replicative capacity declined by an order of magnitude between HBeAgPOS and HBeAgNEG phases of infection. Our estimated lifespan of cccDNA is too short to explain the long durations of chronic infection observed in patients on NA treatment, suggesting that either a sub-population of long-lived hepatocytes harbouring cccDNA molecules persists during therapy, or that NA therapy does not suppress all viral replication. These results provide a greater understanding of the biology of the cccDNA reservoir and can aid the development of new curative therapeutic strategies for treating CHB.

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 knock-down 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.

A PCR assay to quantify patterns of HBV transcription.

Hepatitis B virus (HBV) is the prototype member of the family Hepadnaviridae and replicates via episomal copies of a covalently closed circular DNA (cccDNA) genome of approximately 3.2 kb. The chromatinization of this small viral genome, with overlapping open reading frames and regulatory elements, suggests an important role for epigenetic pathways to regulate HBV transcription. However, the host pathways that regulate HBV transcription and the temporal nature of promoter usage in infected cells are not well understood, in part due to the compact genome structure and overlapping open reading frames. To address this we developed a simple and cost-effective PCR assay to quantify the major viral RNAs and validated this technique using current state-of-art de novo HBV infection model systems. Our PCR method is three orders of magnitude more sensitive than Northern blot and requires relatively small amounts of starting material, making this an attractive tool for assessing HBV transcription.

A Role for B Cells to Transmit Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge on the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We and others have reported that HCV can associate with and infect immune cells and may thereby evade host immune surveillance and elimination. To evaluate whether B cells play a role in HCV transmission, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) HCV patients to infect humanized liver chimeric mice. HCV was transmitted by B cells from chronic infected patients whereas the sera were non-infectious. In contrast, B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. We observed an association between circulating anti-glycoprotein E1E2 antibodies and B cell HCV transmission. Taken together, our studies provide evidence for HCV transmission by B cells, findings that have clinical implications for prophylactic and therapeutic antibody-based vaccine design.

Oxygen Sensing and Viral Replication: Implications for Tropism and Pathogenesis.

The ability to detect and respond to varying oxygen tension is an essential prerequisite to life. Several mechanisms regulate the cellular response to oxygen including the prolyl hydroxylase domain (PHD)/factor inhibiting HIF (FIH)-hypoxia inducible factor (HIF) pathway, cysteamine (2-aminoethanethiol) dioxygenase (ADO) system, and the lysine-specific demethylases (KDM) 5A and KDM6A. Using a systems-based approach we discuss the literature on oxygen sensing pathways in the context of virus replication in different tissues that experience variable oxygen tension. Current information supports a model where the PHD-HIF pathway enhances the replication of viruses infecting tissues under low oxygen, however, the reverse is true for viruses with a selective tropism for higher oxygen environments. Differences in oxygen tension and associated HIF signaling may play an important role in viral tropism and pathogenesis. Thus, pharmaceutical agents that modulate HIF activity could provide novel treatment options for viral infections and associated pathological conditions.

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-h oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components in regulating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, BMAL1 and CLOCK activate rhythmic transcription of genes including the nuclear receptor REV-ERBα, 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. The REV-ERB agonist SR9009 inhibited promoter activity of diverse HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic agonists to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock components in regulating HIV-1 replication.

Hypoxic gene expression in chronic hepatitis B virus infected patients is not observed in state-of-the-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.

Synchronised infection identifies early rate-limiting steps in the hepatitis B virus life cycle.

Hepatitis B virus (HBV) is an enveloped DNA virus that contains a partially double-stranded relaxed circular (rc) DNA. Upon infection, rcDNA is delivered to the nucleus where it is repaired to covalently closed circular (ccc) DNA that serves as the transcription template for all viral RNAs. Our understanding of HBV particle entry dynamics and host pathways regulating intracellular virus trafficking and cccDNA formation is limited. The discovery of sodium taurocholate co-transporting peptide (NTCP) as the primary receptor allows studies on these early steps in viral life cycle. We employed a synchronised infection protocol to quantify HBV entry kinetics. HBV attachment to cells at 4°C is independent of NTCP, however, subsequent particle uptake is NTCP-dependent and reaches saturation at 12 h post-infection. HBV uptake is clathrin- and dynamin dependent with actin and tubulin playing a role in the first 6 h of infection. Cellular fractionation studies demonstrate HBV DNA in the nucleus within 6 h of infection and cccDNA was first detected at 24 h post-infection. Our studies show the majority (83%) of cell bound particles enter HepG2-NTCP cells, however, only a minority (<1%) of intracellular rcDNA was converted to cccDNA, highlighting this as a rate-limiting in establishing infection in vitro. This knowledge highlights the deficiencies in our in vitro cell culture systems and will inform the design and evaluation of physiologically relevant models that support efficient HBV replication.

Hypoxic microenvironment shapes HIV-1 replication and latency.

Viral replication is defined by the cellular microenvironment and one key factor is local oxygen tension, where hypoxia inducible factors (HIFs) regulate the cellular response to oxygen. Human immunodeficiency virus (HIV) infected cells within secondary lymphoid tissues exist in a low-oxygen or hypoxic environment in vivo. However, the majority of studies on HIV replication and latency are performed under laboratory conditions where HIFs are inactive. We show a role for HIF-2α in restricting HIV transcription via direct binding to the viral promoter. Hypoxia reduced tumor necrosis factor or histone deacetylase inhibitor, Romidepsin, mediated reactivation of HIV and inhibiting HIF signaling-pathways reversed this phenotype. Our data support a model where the low-oxygen environment of the lymph node may suppress HIV replication and promote latency. We identify a mechanism that may contribute to the limited efficacy of latency reversing agents in reactivating HIV and suggest new strategies to control latent HIV-1.

Accurate targeted long-read DNA methylation and hydroxymethylation sequencing with TAPS.

We present long-read Tet-assisted pyridine borane sequencing (lrTAPS) for targeted base-resolution sequencing of DNA methylation and hydroxymethylation in regions up to 10 kb from nanogram-level input. Compatible with both Oxford Nanopore and PacBio Single-Molecule Real-Time (SMRT) sequencing, lrTAPS detects methylation with accuracy comparable to short-read Illumina sequencing but with long-range epigenetic phasing. We applied lrTAPS to sequence difficult-to-map regions in mouse embryonic stem cells and to identify distinct methylation events in the integrated hepatitis B virus genome.

Bacterial flagellin promotes viral entry via an NF-kB and Toll Like Receptor 5 dependent pathway.

Viruses and bacteria colonize hosts by invading epithelial barriers. Recent studies have shown that interactions between the microbiota, pathogens and the host can potentiate infection through poorly understood mechanisms. Here, we investigated whether diverse bacterial species could modulate virus internalization into host cells, often a rate-limiting step in establishing infections. Lentiviral pseudoviruses expressing influenza, measles, Ebola, Lassa or vesicular stomatitis virus envelope glycoproteins enabled us to study entry of viruses that exploit diverse internalization pathways. Salmonella Typhimurium, Escherichia coli and Pseudomonas aeruginosa significantly increased viral uptake, even at low bacterial frequencies. This did not require bacterial contact with or invasion of host cells. Studies determined that the bacterial antigen responsible for this pro-viral activity was the Toll-Like Receptor 5 (TLR5) agonist flagellin. Exposure to flagellin increased virus attachment to epithelial cells in a temperature-dependent manner via TLR5-dependent activation of NF-ΚB. Importantly, this phenotype was both long lasting and detectable at low multiplicities of infection. Flagellin is shed from bacteria and our studies uncover a new bystander role for this protein in regulating virus entry. This highlights a new aspect of viral-bacterial interplay with significant implications for our understanding of polymicrobial-associated pathogenesis.

A dual role for SAMHD1 in regulating HBV cccDNA and RT-dependent particle genesis.

Chronic hepatitis B is one of the world's unconquered diseases with more than 240 million infected subjects at risk of developing liver disease and hepatocellular carcinoma. Hepatitis B virus reverse transcribes pre-genomic RNA to relaxed circular DNA (rcDNA) that comprises the infectious particle. To establish infection of a naïve target cell, the newly imported rcDNA is repaired by host enzymes to generate covalently closed circular DNA (cccDNA), which forms the transcriptional template for viral replication. SAMHD1 is a component of the innate immune system that regulates deoxyribonucleoside triphosphate levels required for host and viral DNA synthesis. Here, we show a positive role for SAMHD1 in regulating cccDNA formation, where KO of SAMHD1 significantly reduces cccDNA levels that was reversed by expressing wild-type but not a mutated SAMHD1 lacking the nuclear localization signal. The limited pool of cccDNA in infected Samhd1 KO cells is transcriptionally active, and we observed a 10-fold increase in newly synthesized rcDNA-containing particles, demonstrating a dual role for SAMHD1 to both facilitate cccDNA genesis and to restrict reverse transcriptase-dependent particle genesis.

Hepatitis C virus infection is associated with hepatic and adipose tissue insulin resistance that improves after viral cure.

BACKGROUND: Chronic hepatitis C (CHC) is associated with systemic insulin resistance, yet there are limited data on the tissue-specific contribution in vivo to this adverse metabolic phenotype, and the effect of HCV cure. METHODS: We examined tissue-specific insulin sensitivity in a cohort study involving 13 patients with CHC compared to 12 BMI-matched healthy control subjects. All subjects underwent a two-step clamp incorporating the use of stable isotopes to measure carbohydrate and lipid flux (hepatic and global insulin sensitivity) with concomitant subcutaneous adipose tissue microdialysis and biopsy (subcutaneous adipose tissue insulin sensitivity). Investigations were repeated in seven patients with CHC following antiviral therapy with a documented sustained virological response. RESULTS: Adipose tissue was more insulin resistant in patients with CHC compared to healthy controls, as evidence by elevated glycerol production rate and impaired insulin-mediated suppression of both circulating nonesterified fatty acids (NEFA) and adipose interstitial fluid glycerol release during the hyperinsulinaemic euglycaemic clamp. Hepatic and muscle insulin sensitivity were similar between patients with CHC and controls. Following viral eradication, hepatic insulin sensitivity improved as demonstrated by a reduction in endogenous glucose production rate. In addition, circulating NEFA decreased with sustained virological response (SVR) and insulin was more effective at suppressing adipose tissue interstitial glycerol release with a parallel increase in the expression of insulin signalling cascade genes in adipose tissue consistent with enhanced adipose tissue insulin sensitivity. CONCLUSION: Chronic hepatitis C patients have profound subcutaneous adipose tissue insulin resistance in comparison with BMI-matched controls. For the first time, we have demonstrated that viral eradication improves global, hepatic and adipose tissue insulin sensitivity.