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.

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.

Pseudouridine (Ψ) 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. Here, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS), which enables Ψ-to-C transitions, for quantitative profiling of Ψ at single-base resolution. BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS detected all known Ψ sites in human rRNA and spliceosomal small nuclear RNAs and generated the quantitative Ψ map of human small nucleolar RNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine editing sites and N¹-methyladenosine. Depletion of pseudouridine synthases TRUB1, PUS7 and PUS1 elucidated their targets and sequence motifs. We further identified a highly abundant Ψ₁₁₄ site in Epstein–Barr virus-encoded small RNA EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation across virus families.

The coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (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 to angiotensin-converting enzyme (ACE2). Circadian rhythms coordinate an organism's response to its environment and can regulate host susceptibility to virus infection. We demonstrate that silencing the circadian regulator Bmal1 or treating lung epithelial cells with the REV-ERB agonist SR9009 reduces ACE2 expression and inhibits SARS-CoV-2 entry and replication. Importantly, treating infected cells with SR9009 limits SARS-CoV-2 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 interferon-stimulated gene transcripts in Calu-3 lung epithelial cells, providing a mechanism for the circadian pathway to limit SARS-CoV-2 infection. Our study highlights alternative approaches to understand and improve therapeutic targeting of SARS-CoV-2.

Hypoxia inducible factors (HIFs) are a hallmark of inflammation and are key regulators of hepatic immunity and metabolism, yet their role in HBV replication is poorly defined. HBV replicates in hepatocytes within the liver, a naturally hypoxic organ, however most studies of viral replication are performed under conditions of atmospheric oxygen, where HIFs are inactive. We therefore investigated the role of HIFs in regulating HBV replication. Using cell culture, animal models, human tissue and pharmacological agents inhibiting the HIF-prolyl hydroxylases, we investigated the impact of hypoxia on the HBV life cycle. Culturing liver cell-based model systems under low oxygen uncovered a new role for HIFs in binding HBV DNA and activating the basal core promoter, leading to increased pre-genomic RNA and de novo HBV particle secretion. The presence of hypoxia responsive elements among all primate members of the hepadnaviridae highlights an evolutionary conserved role for HIFs in regulating this virus family. Identifying a role for this conserved oxygen sensor in regulating HBV transcription suggests that this virus has evolved to exploit the HIF signaling pathway to persist in the low oxygen environment of the liver. Our studies show the importance of considering oxygen availability when studying HBV-host interactions and provide innovative routes to better understand and target chronic HBV infection. Viral replication in host cells is defined by the cellular microenvironment and one key factor is local oxygen tension. Hepatitis B virus (HBV) replicates in the liver, a naturally hypoxic organ. Hypoxia inducible factors (HIFs) are the major sensors of low oxygen; herein, we identify a new role for these factors in regulating HBV replication, revealing new therapeutic targets.

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.