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.

Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.

SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses.

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patients on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Emx2 underlies the development and evolution of marsupial gliding membranes.

Phenotypic variation among species is a product of evolutionary changes to developmental programs1,2. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.

Mathematical Models of Early Hepatitis B Virus Dynamics in Humanized Mice.

Analyzing the impact of the adaptive immune response during acute hepatitis B virus (HBV) infection is essential for understanding disease progression and control. Here we developed mathematical models of HBV infection which either lack terms for adaptive immune responses, or assume adaptive immune responses in the form of cytolytic immune killing, non-cytolytic immune cure, or non-cytolytic-mediated block of viral production. We validated the model that does not include immune responses against temporal serum hepatitis B DNA (sHBV) and temporal serum hepatitis B surface-antigen (HBsAg) experimental data from mice engrafted with human hepatocytes (HEP). Moreover, we validated the immune models against sHBV and HBsAg experimental data from mice engrafted with HEP and human immune system (HEP/HIS). As expected, the model that does not include adaptive immune responses matches the observed high sHBV and HBsAg concentrations in all HEP mice. By contrast, while all immune response models predict reduction in sHBV and HBsAg concentrations in HEP/HIS mice, the Akaike Information Criterion cannot discriminate between non-cytolytic cure (resulting in a class of cells refractory to reinfection) and antiviral block functions (of up to 99 % viral production 1-3 weeks following peak viral load). We can, however, reject cytolytic killing, as it can only match the sHBV and HBsAg data when we predict unrealistic levels of hepatocyte loss.

Mechanisms of Hepatitis B Virus cccDNA and Minichromosome Formation and HBV Gene Transcription.

Hepatitis B virus (HBV) is the etiologic agent of chronic hepatitis B, which puts at least 300 million patients at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. HBV is a partially double-stranded DNA virus of the Hepadnaviridae family. While HBV was discovered more than 50 years ago, many aspects of its replicative cycle remain incompletely understood. Central to HBV persistence is the formation of covalently closed circular DNA (cccDNA) from the incoming relaxed circular DNA (rcDNA) genome. cccDNA persists as a chromatinized minichromosome and is the major template for HBV gene transcription. Here, we review how cccDNA and the viral minichromosome are formed and how viral gene transcription is regulated and highlight open questions in this area of research.

The impact of epitranscriptomic modifications on liver disease.

RNA modifications have emerged as important mechanisms of gene regulation. Developmental, metabolic, and cell cycle regulatory processes are all affected by epitranscriptomic modifications, which control gene expression in a dynamic manner. The hepatic tissue is highly metabolically active and has an impressive ability to regenerate after injury. Cell proliferation, differentiation, and metabolism, which are all essential to the liver response to injury and regeneration, are regulated via RNA modification. Two such modifications, N6-methyladenosine (m6A)and 5-methylcytosine (m5C), have been identified as prognostic disease markers and potential therapeutic targets for liver diseases. Here, we describe progress in understanding the role of RNA modifications in liver biology and disease and discuss specific areas where unexpected results could lead to improved future understanding.

Membrane-proximal motifs encode differences in signaling strength between type I and III interferon receptors.

Interferons (IFNs) play crucial roles in antiviral defenses. Despite using the same Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) signaling cascade, type I and III IFN receptors differ in the magnitude and dynamics of their signaling in terms of STAT phosphorylation, gene transcription, and antiviral responses. These differences are not due to ligand-binding affinity and receptor abundance. Here, we investigated the ability of the intracellular domains (ICDs) of IFN receptors to differentiate between type I and III IFN signaling. We engineered synthetic, heterodimeric type I and III IFN receptors that were stably expressed at similar amounts in human cells and responded to a common ligand. We found that our synthetic type I IFN receptors stimulated STAT phosphorylation and gene expression to greater extents than did the corresponding type III IFN receptors. Furthermore, we identified short "box motifs" within ICDs that bind to JAK1 that were sufficient to encode differences between the type I and III IFN receptors. Together, our results indicate that specific regions within the ICDs of IFN receptor subunits encode different downstream signaling strengths that enable type I and III IFN receptors to produce distinct signaling outcomes.

A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns.

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we study the coat of the African striped mouse (Rhabdomys pumilio) to uncover how periodic stripes form. Combining transcriptomics, mathematical modelling and mouse transgenics, we show that the Wnt modulator Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing in vivo gene editing in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants exhibited changes in pigmentation, revealing that Sfrp2 also regulates hair colour. Lastly, through evolutionary analyses, we find that striped mice have evolved lineage-specific changes in regulatory elements surrounding Sfrp2, many of which may be implicated in modulating the expression of this gene. Altogether, our results show that a single factor controls coat pattern formation by acting both as an orienting signalling mechanism and a modulator of pigmentation. More broadly, our work provides insights into how spatial patterns are established in developing embryos and the mechanisms by which phenotypic novelty originates.

Synthetic Heterodimers of Type III Interferon Receptors Require TYK2 for STAT Activation.

Type III interferons (IFN-λ) are central to host defense against viral infection of epithelial barrier surfaces. IFN-λ binding to its receptor induces a JAK-STAT cascade through kinases Janus-associated kinase 1 (JAK1) and tyrosine kinase 2 (TYK2), which are associated on either subunit of the heterodimeric type III IFN receptor. Recent studies have shown that TYK2 is not necessary for IFN-λ to signal, in contrast to IFN-α, which uses the same JAK-STAT pathway activated by the type I IFN receptor. The mechanism for this differential TYK2 requirement is unknown. Our study uses synthetic IFN receptors in TYK2-deficient U2OS epithelial cells to define the processes in type I and III IFN signaling that require TYK2. We find that TYK2 deficiency reduces signaling equally from heterodimers of either type I or III IFN receptor intracellular domains. In contrast, JAK1-associated homodimers of IFNAR2 or IFNLR1 are both fully signaling competent even in the absence of TYK2. These results suggest that heterodimerization of the type III IFN receptor is insufficient to confer TYK2-independent signaling. Thus, we propose that noncanonical receptor complexes may participate in endogenous type III IFN signaling to confer TYK2-independent signaling downstream of IFN-λ stimulation.

Highly multiplexed mRNA quantitation with CRISPR-Cas13.

RNA quantitation tools are often either high-throughput or cost-effective, but rarely are they both. Existing methods can profile the transcriptome at great expense or are limited to quantifying a handful of genes by labor constraints. A technique that permits more throughput at a reduced cost could enable multi-gene kinetic studies, gene regulatory network analysis, and combinatorial genetic screens. Here, we introduce quantitative Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (qCARMEN): an RNA quantitation technique which leverages the programmable RNA-targeting capabilities of CRISPR-Cas13 to address this challenge by quantifying over 4,500 gene-sample pairs in a single experiment. Using qCARMEN, we studied the response profiles of interferon-stimulated genes (ISGs) during interferon (IFN) stimulation and flavivirus infection. Additionally, we observed isoform switching kinetics during epithelial-mesenchymal transition. qCARMEN is a simple and inexpensive technique that greatly enhances the scalability of RNA quantitation for novel applications with performance similar to gold-standard methods.

Persistent hepatitis B virus and HIV coinfections in dually humanized mice engrafted with human liver and immune system.

Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including human immunodeficiency virus type (HIV) and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV, which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV, which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection, and preclinical testing of novel immunotherapeutics.

Hepatitis delta virus RNA decline post-inoculation in human NTCP transgenic mice is biphasic.

Chronic infection with hepatitis B and delta viruses (HDV) is the most serious form of viral hepatitis due to more severe manifestations of an accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. We characterized early HDV kinetics post-inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor-human sodium taurocholate co-transporting polypeptide (hNTCP). Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific-binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 35 minutes (standard error, SE: 6.3), binds to non-specific cells with a rate of 0.05 per hour (SE: 0.01), and returns as free virus with a rate of 0.11 per hour (SE: 0.02). Characterizing early HDV-host kinetics elucidates how quickly HDV is either cleared or bound depending on the immunological background and hNTCP presence. IMPORTANCE The persistence phase of HDV infection has been studied in some animal models; however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post-inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics.

Targeted viral adaptation generates a simian-tropic hepatitis B virus that infects marmoset cells.

Hepatitis B virus (HBV) only infects humans and chimpanzees, posing major challenges for modeling HBV infection and chronic viral hepatitis. The major barrier in establishing HBV infection in non-human primates lies at incompatibilities between HBV and simian orthologues of the HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Through mutagenesis analysis and screening among NTCP orthologues from Old World monkeys, New World monkeys and prosimians, we determined key residues responsible for viral binding and internalization, respectively and identified marmosets as a suitable candidate for HBV infection. Primary marmoset hepatocytes and induced pluripotent stem cell-derived hepatocyte-like cells support HBV and more efficient woolly monkey HBV (WMHBV) infection. Adapted chimeric HBV genome harboring residues 1-48 of WMHBV preS1 generated here led to a more efficient infection than wild-type HBV in primary and stem cell derived marmoset hepatocytes. Collectively, our data demonstrate that minimal targeted simianization of HBV can break the species barrier in small NHPs, paving the path for an HBV primate model.

Hepatitis delta virus RNA decline post inoculation in human NTCP transgenic mice is biphasic.

Background and Aims: Chronic infection with hepatitis B and hepatitis delta viruses (HDV) is considered the most serious form of viral hepatitis due to more severe manifestations of and accelerated progression to liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no FDA-approved treatment for HDV and current interferon-alpha treatment is suboptimal. We characterized early HDV kinetics post inoculation and incorporated mathematical modeling to provide insights into host-HDV dynamics. Methods: We analyzed HDV RNA serum viremia in 192 immunocompetent (C57BL/6) and immunodeficient (NRG) mice that did or did not transgenically express the HDV receptor - human sodium taurocholate co-transporting peptide (hNTCP). Results: Kinetic analysis indicates an unanticipated biphasic decline consisting of a sharp first-phase and slower second-phase decline regardless of immunocompetence. HDV decline after re-inoculation again followed a biphasic decline; however, a steeper second-phase HDV decline was observed in NRG-hNTCP mice compared to NRG mice. HDV-entry inhibitor bulevirtide administration and HDV re-inoculation indicated that viral entry and receptor saturation are not major contributors to clearance, respectively. The biphasic kinetics can be mathematically modeled by assuming the existence of a non-specific binding compartment with a constant on/off-rate and the steeper second-phase decline by a loss of bound virus that cannot be returned as free virus to circulation. The model predicts that free HDV is cleared with a half-life of 18 minutes (standard error, SE: 2.4), binds to non-specific cells with a rate of 0.06 hour -1 (SE: 0.03), and returns as free virus with a rate of 0.23 hour -1 (SE: 0.03). Conclusions: Understanding early HDV-host kinetics will inform pre-clinical therapeutic kinetic studies on how the efficacy of anti-HDV therapeutics can be affected by early kinetics of viral decline. LAY SUMMARY: The persistence phase of HDV infection has been studied in some animal models, however, the early kinetics of HDV in vivo is incompletely understood. In this study, we characterize an unexpectedly HDV biphasic decline post inoculation in immunocompetent and immunodeficient mouse models and use mathematical modeling to provide insights into HDV-host dynamics. Understanding the kinetics of viral clearance in the blood can aid pre-clinical development and testing models for anti-HDV therapeutics.

Structural features stabilized by divalent cation coordination within hepatitis E virus ORF1 are critical for viral replication.

Hepatitis E virus (HEV) is an RNA virus responsible for over 20 million infections annually. HEV's open reading frame (ORF)1 polyprotein is essential for genome replication, though it is unknown how the different subdomains function within a structural context. Our data show that ORF1 operates as a multifunctional protein, which is not subject to proteolytic processing. Supporting this model, scanning mutagenesis performed on the putative papain-like cysteine protease (pPCP) domain revealed six cysteines essential for viral replication. Our data are consistent with their role in divalent metal ion coordination, which governs local and interdomain interactions that are critical for the overall structure of ORF1; furthermore, the 'pPCP' domain can only rescue viral genome replication in trans when expressed in the context of the full-length ORF1 protein but not as an individual subdomain. Taken together, our work provides a comprehensive model of the structure and function of HEV ORF1.

Barriers to hepatitis C virus infection in mice.

Hepatitis C virus (HCV) is unable to infect mice, a fact that has severely limited their use as small-animal models for HCV pathogenesis and as tools for HCV vaccine development. HCV is blocked at various stages of its life cycle in mouse cells, due to incompatibility with host factors, the presence of dominant restriction factors, and effective immune responses. Molecular mechanisms for several such blocks have been characterized. The stepwise understanding of these limitations in mice will enable the development of an immunocompetent mouse that can fully support HCV infection and exhibit disease similar to that of infected humans.

Photochemical Identification of Auxiliary Severe Acute Respiratory Syndrome Coronavirus 2 Host Entry Factors Using µMap.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform µMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes µMap as a powerful tool for rapidly interrogating host-virus interactomes.