Highlights from the 2023 International Meeting on the Molecular Biology of Hepatitis B virus.

Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.

Oxygen-dependent histone lysine demethylase 4 restricts hepatitis B virus replication.

Mammalian cells have evolved strategies to regulate gene expression when oxygen is limited. Hypoxia-inducible factors (HIF) are the major transcriptional regulators of host gene expression. We previously reported that HIFs bind and activate hepatitis B virus (HBV) DNA transcription under low oxygen conditions; however, the global cellular response to low oxygen is mediated by a family of oxygenases that work in concert with HIFs. Recent studies have identified a role for chromatin modifiers in sensing cellular oxygen and orchestrating transcriptional responses, but their role in the HBV life cycle is as yet undefined. We demonstrated that histone lysine demethylase 4 (KDM4) can restrict HBV, and pharmacological or oxygen-mediated inhibition of the demethylase increases viral RNAs derived from both episomal and integrated copies of the viral genome. Sequencing studies demonstrated that KDM4 is a major regulator of the hepatic transcriptome, which defines hepatocellular permissivity to HBV infection. We propose a model where HBV exploits cellular oxygen sensors to replicate and persist in the liver. Understanding oxygen-dependent pathways that regulate HBV infection will facilitate the development of physiologically relevant cell-based models that support efficient HBV replication.

CTCF regulates hepatitis B virus cccDNA chromatin topology.

Hepatitis B Virus (HBV) is a small DNA virus that replicates via an episomal covalently closed circular DNA (cccDNA) that serves as the transcriptional template for viral mRNAs. The host protein, CCCTC-binding factor (CTCF), is a key regulator of cellular transcription by maintaining epigenetic boundaries, nucleosome phasing, stabilisation of long-range chromatin loops and directing alternative exon splicing. We previously reported that CTCF binds two conserved motifs within Enhancer I of the HBV genome and represses viral transcription, however, the underlying mechanisms were not identified. We show that CTCF depletion in cells harbouring cccDNA-like HBV molecules and in de novo infected cells resulted in an increase in spliced transcripts, which was most notable in the abundant SP1 spliced transcript. In contrast, depletion of CTCF in cell lines with integrated HBV DNA had no effect on the abundance of viral transcripts and in line with this observation there was limited evidence for CTCF binding to viral integrants, suggesting that CTCF-regulation of HBV transcription is specific to episomal cccDNA. Analysis of HBV chromatin topology by Assay for Transposase Accessible Chromatin Sequencing (ATAC-Seq) revealed an accessible region spanning Enhancers I and II and the basal core promoter (BCP). Mutating the CTCF binding sites within Enhancer I resulted in a dramatic rearrangement of chromatin accessibility where the open chromatin region was no longer detected, indicating loss of the phased nucleosome up- and down-stream of the HBV enhancer/BCP. These data demonstrate that CTCF functions to regulate HBV chromatin conformation and nucleosomal positioning in episomal maintained cccDNA, which has important consequences for HBV transcription regulation.

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. Our knowledge of the host pathways that define susceptibility to infection and disease severity are limited. Hypoxia inducible factors (HIFs) define metabolic responses to low oxygen and regulate inflammatory responses in the lower respiratory tract. We demonstrate a role for HIFs to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors reduce the expression of the RSV entry receptor nucleolin and inhibit viral 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 this respiratory pathogen.

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 (BCP). Here we show that the hypoxic-dependent increase in BCP-derived transcripts is dependent on N6-methyladenosine (m6A) modifications in the 5' stem loop that regulate RNA half-life. Application of a probe-enriched long-read sequencing method to accurately map the HBV transcriptome showed an increased abundance of pre-genomic RNA under hypoxic conditions. Mapping the transcription start sites of BCP-RNAs identified a role for hypoxia to regulate pre-genomic RNA splicing that is dependent on m6A modification. Bioinformatic 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-NTCP cell line showed increased ALKBH5 gene expression under hypoxic conditions and a concomitant reduction in m6A-modified HBV BCP-RNA and host RNAs. Silencing the demethylase reduced the level of BCP-RNAs and host gene (CA9, NDRG1, VEGFA, BNIP3, FUT11, GAP and P4HA1) transcripts and this was mediated via reduced HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

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 bioinformatic 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 HIFα expression. In summary, our study highlights a previously unrecognized role for ALKBH5 in orchestrating viral and cellular transcriptional responses to low oxygen.

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.

Inhibition of salt inducible kinases reduces rhythmic HIV-1 replication and reactivation from latency.

Human immunodeficiency virus type 1 (HIV-1) causes a major burden on global health, and eradication of latent virus infection is one of the biggest challenges in the field. The circadian clock is an endogenous timing system that oscillates with a ~24 h period regulating multiple physiological processes and cellular functions, and we recently reported that the cell intrinsic clock regulates rhythmic HIV-1 replication. Salt inducible kinases (SIK) contribute to circadian regulatory networks, however, there is limited evidence for SIKs regulating HIV-1 infection. Here, we show that pharmacological inhibition of SIKs perturbed the cellular clock and reduced rhythmic HIV-1 replication in circadian synchronised cells. Further, SIK inhibitors or genetic silencing of Sik expression inhibited viral replication in primary cells and in a latency model, respectively. Overall, this study demonstrates a role for salt inducible kinases in regulating HIV-1 replication and latency reactivation, which can provide innovative routes to better understand and target latent HIV-1 infection.

Molecular components of the circadian clock regulate HIV-1 replication.

Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism's response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

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.

HLA-E-restricted SARS-CoV-2-specific T cells from convalescent COVID-19 patients suppress virus replication despite HLA class Ia down-regulation.

Pathogen-specific CD8+ T cell responses restricted by the nonpolymorphic nonclassical class Ib molecule human leukocyte antigen E (HLA-E) are rarely reported in viral infections. The natural HLA-E ligand is a signal peptide derived from classical class Ia HLA molecules that interact with the NKG2/CD94 receptors to regulate natural killer cell functions, but pathogen-derived peptides can also be presented by HLA-E. Here, we describe five peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that elicited HLA-E-restricted CD8+ T cell responses in convalescent patients with coronavirus disease 2019. These T cell responses were identified in the blood at frequencies similar to those reported for classical HLA-Ia-restricted anti-SARS-CoV-2 CD8+ T cells. HLA-E peptide-specific CD8+ T cell clones, which expressed diverse T cell receptors, suppressed SARS-CoV-2 replication in Calu-3 human lung epithelial cells. SARS-CoV-2 infection markedly down-regulated classical HLA class I expression in Calu-3 cells and primary reconstituted human airway epithelial cells, whereas HLA-E expression was not affected, enabling T cell recognition. Thus, HLA-E-restricted T cells could contribute to the control of SARS-CoV-2 infection alongside classical T cells.

An enrichment protocol and analysis pipeline for long read sequencing of the hepatitis B virus transcriptome.

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.

An ACAT inhibitor suppresses SARS-CoV-2 replication and boosts 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 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 pseudoparticle infection and disrupts the association of ACE2 and GM1 lipid rafts on the cell membrane, perturbing viral attachment. Imaging SARS-CoV-2 RNAs at the single cell level using a viral replicon model identifies the capacity of Avasimibe to limit the establishment of replication complexes required for RNA replication. Genetic studies to transiently silence or overexpress ACAT isoforms confirmed a role for ACAT in SARS-CoV-2 infection. Furthermore, Avasimibe boosts the expansion of functional SARS-CoV-2-specific T cells from the blood of patients sampled during the acute phase of infection. Thus, re-purposing of ACAT inhibitors provides a compelling therapeutic strategy for the treatment of COVID-19 to achieve both antiviral and immunomodulatory effects. Trial registration: NCT04318314.

Sleep and circadian rhythm disruption alters the lung transcriptome to predispose to viral infection.

Sleep and circadian rhythm disruption (SCRD), as encountered during shift work, increases the risk of respiratory viral infection including SARS-CoV-2. However, the mechanism(s) underpinning higher rates of respiratory viral infection following SCRD remain poorly characterized. To address this, we investigated the effects of acute sleep deprivation on the mouse lung transcriptome. Here we show that sleep deprivation profoundly alters the transcriptional landscape of the lung, causing the suppression of both innate and adaptive immune systems, disrupting the circadian clock, and activating genes implicated in SARS-CoV-2 replication, thereby generating a lung environment that could promote viral infection and associated disease pathogenesis. Our study provides a mechanistic explanation of how SCRD increases the risk of respiratory viral infections including SARS-CoV-2 and highlights possible therapeutic avenues for the prevention and treatment of respiratory viral infection.

Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.

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 Severe-acute-respiratory-syndrome-coronavirus-2 (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 the Syrian hamster model. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced 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.

Time-of-Day Variation in SARS-CoV-2 RNA Levels during the Second Wave of COVID-19.

Circadian rhythms influence and coordinate an organism's response to its environment and to invading pathogens. We studied the diurnal variation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasal/throat swabs collected in late 2020 to spring 2021 in a population immunologically naïve to SARS-CoV-2 and prior to widespread vaccination. SARS-CoV-2 diagnostic PCR data from 1698 participants showed a significantly higher viral load in samples obtained in the afternoon, in males, and in hospitalised patients when linear mixed modelling was applied. This study illustrates the importance of recording sample collection times when measuring viral replication parameters in clinical and research studies.

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease.

Chronic hepatitis B virus (HBV) infection is a global health problem that presents as a spectrum of liver disease, reflecting an interplay between the virus and the host immune system. HBV genomes exist as episomal covalently closed circular DNA (cccDNA) or chromosomal integrants. The relative contribution of these genomes to the viral transcriptome in chronic hepatitis B (CHB) is not well-understood. We developed a qPCR method to estimate the abundance of HBV cccDNA- and integrant-derived viral transcripts and applied this to a cohort of patients diagnosed with CHB in the HBe antigen negative phase of disease. We noted a variable pattern of HBV transcripts from both DNA templates, with preS1/S2 mRNAs predominating and a significant association between increasing age and the expression of integrant-derived mRNAs, but not with inflammatory status. In contrast, cccDNA-derived transcripts were associated with markers of liver inflammation. Analysis of the inflammatory hepatic transcriptome identified 24 genes significantly associated with cccDNA transcriptional activity. Our study uncovers an immune gene signature that associates with HBV cccDNA transcription and increases our understanding of viral persistence.

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.