RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Comparative analysis of human, rodent and snake deltavirus replication.

The recent discovery of Hepatitis D (HDV)-like viruses across a wide range of taxa led to the establishment of the Kolmioviridae family. Recent studies suggest that kolmiovirids can be satellites of viruses other than Hepatitis B virus (HBV), challenging the strict HBV/HDV-association dogma. Studying whether kolmiovirids are able to replicate in any animal cell they enter is essential to assess their zoonotic potential. Here, we compared replication of three kolmiovirids: HDV, rodent (RDeV) and snake (SDeV) deltavirus in vitro and in vivo. We show that SDeV has the narrowest and RDeV the broadest host cell range. High resolution imaging of cells persistently replicating these viruses revealed nuclear viral hubs with a peculiar RNA-protein organization. Finally, in vivo hydrodynamic delivery of viral replicons showed that both HDV and RDeV, but not SDeV, efficiently replicate in mouse liver, forming massive nuclear viral hubs. Our comparative analysis lays the foundation for the discovery of specific host factors controlling Kolmioviridae host-shifting.

Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8+ T Cells in Chronic versus Acute Infection.

Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

A human liver cell atlas reveals heterogeneity and epithelial progenitors.

The human liver is an essential multifunctional organ. The incidence of liver diseases is rising and there are limited treatment options. However, the cellular composition of the liver remains poorly understood. Here we performed single-cell RNA sequencing of about 10,000 cells from normal liver tissue from nine human donors to construct a human liver cell atlas. Our analysis identified previously unknown subtypes of endothelial cells, Kupffer cells, and hepatocytes, with transcriptome-wide zonation of some of these populations. We show that the EPCAM+ population is heterogeneous, comprising hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As a proof-of-principle, we used our atlas to unravel the phenotypic changes that occur in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource to enable the discovery of previously unknown cell types in normal and diseased livers.

Tight Junction Proteins as Therapeutic Targets to Treat Liver Fibrosis and Hepatocellular Carcinoma.

In the last decade tight junction proteins exposed at the surface of liver or cancer cells have been uncovered as mediators of liver disease biology: Claudin-1 and Occludin are host factors for hepatitis C virus entry and Claudin-1 has been identified as a driver for liver fibrosis and hepatocellular carcinoma (HCC). Moreover, Claudins have emerged as therapeutic targets for liver disease and HCC. CLDN1 expression is upregulated in liver fibrosis and HCC. Monoclonal antibodies (mAbs) targeting Claudin-1 have completed preclinical proof-of-concept studies for treatment of liver fibrosis and HCC and are currently in clinical development for advanced liver fibrosis. Claudin-6 overexpression is associated with an HCC aggressive phenotype and treatment resistance. Claudin-6 mAbs or chimeric antigen receptor-T cells therapies are currently being clinically investigated for Claudin-6 overexpressing tumors. In conclusion, targeting Claudin proteins offers a novel clinical opportunity for the treatment of patients with advanced liver fibrosis and HCC.

JAK1 promotes HDV replication and is a potential target for antiviral therapy.

BACKGROUND & AIMS: Chronic co-infection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. To date, no treatment induces efficient viral clearance, and a better characterization of virus-host interactions is required to develop new therapeutic strategies. METHODS: Using loss-of-function strategies, we validated the unexpected proviral activity of Janus kinase 1 (JAK1) - a key player in innate immunity - in the HDV life cycle and determined its mechanism of action on HDV through various functional analyses including co-immunoprecipitation assays. RESULTS: We confirmed the key role of JAK1 kinase activity in HDV infection. Moreover, our results suggest that JAK1 inhibition is associated with a modulation of ERK1/2 activation and S-HDAg phosphorylation, which is crucial for viral replication. Finally, we showed that FDA-approved JAK1-specific inhibitors are efficient antivirals in relevant in vitro models including primary human hepatocytes. CONCLUSIONS: Taken together, we uncovered JAK1 as a key host factor for HDV replication and a potential target for new antiviral treatment. IMPACT AND IMPLICATIONS: Chronic hepatitis D is the most aggressive form of chronic viral hepatitis. As no curative treatment is currently available, new therapeutic strategies based on host-targeting agents are urgently needed. Here, using loss-of-function strategies, we uncover an unexpected interaction between JAK1, a major player in the innate antiviral response, and HDV infection. We demonstrated that JAK1 kinase activity is crucial for both the phosphorylation of the delta antigen and the replication of the virus. By demonstrating the antiviral potential of several FDA-approved JAK1 inhibitors, our results could pave the way for the development of innovative therapeutic strategies to tackle this global health threat.

Targeting the liver clock improves fibrosis by restoring TGF-ß signaling.

BACKGROUND & AIMS: Liver fibrosis is the major driver of hepatocellular carcinoma and liver disease-related death. Approved antifibrotic therapies are absent and compounds in development have limited efficacy. Increased TGF-ß signaling drives collagen deposition by hepatic stellate cells (HSCs)/myofibroblasts. Here, we aimed to dissect the role of the circadian clock (CC) in controlling TGF-ß signaling and liver fibrosis. METHODS: Using CC-mutant mice, enriched HSCs and myofibroblasts obtained from healthy and fibrotic mice in different CC phases and loss-of-function studies in human hepatocytes and myofibroblasts, we investigated the relationship between CC and TGF-ß signaling. We explored hepatocyte-myofibroblast communication through bioinformatic analyses of single-nuclei transcriptomes and performed validation in cell-based models. Using mouse models for MASH (metabolic dysfunction-associated steatohepatitis)-related fibrosis and spheroids from patients with liver disease, we performed proof-of-concept studies to validate pharmacological targetability and clinical translatability. RESULTS: We discovered that the CC oscillator temporally gates TGF-ß signaling and this regulation is broken in fibrosis. We demonstrate that HSCs and myofibroblasts contain a functional CC with rhythmic expression of numerous genes, including fibrogenic genes. Perturbation studies in hepatocytes and myofibroblasts revealed a reciprocal relationship between TGF-ß activation and CC perturbation, which was confirmed in patient-derived ex vivo and in vivo models. Pharmacological modulation of CC-TGF-ß signaling inhibited fibrosis in mouse models in vivo as well as in patient-derived liver spheroids. CONCLUSION: The CC regulates TGF-ß signaling, and the breakdown of this control is associated with liver fibrosis in patients. Pharmacological proof-of-concept studies across different models have uncovered the CC as a novel therapeutic target for liver fibrosis - a growing unmet medical need. IMPACT AND IMPLICATIONS: Liver fibrosis due to metabolic diseases is a global health challenge. Many liver functions are rhythmic throughout the day, being controlled by the circadian clock (CC). Here we demonstrate that regulation of the CC is perturbed upon chronic liver injury and this perturbation contributes to fibrotic disease. By showing that a compound targeting the CC improves liver fibrosis in patient-derived models, this study provides a novel therapeutic candidate strategy to treat fibrosis in patients. Additional studies will be needed for clinical translation. Since the findings uncover a previously undiscovered profibrotic mechanism and therapeutic target, the study is of interest for scientists investigating liver disease, clinical hepatologists and drug developers.

IL-22 regulates MASTL expression in intestinal epithelial cells.

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.

Novel non-HAP class A HBV capsid assembly modulators have distinct in vitro and in vivo profiles.

IMPORTANCE: Chronic hepatitis B is the most important cause of liver cancer worldwide and affects more than 290 million people. Current treatments are mostly suppressive and rarely lead to a cure. Therefore, there is a need for novel and curative drugs that target the host or the causative agent, hepatitis B virus itself. Capsid assembly modulators are an interesting class of antiviral molecules that may one day become part of curative treatment regimens for chronic hepatitis B. Here we explore the characteristics of a particularly interesting subclass of capsid assembly modulators. These so-called non-HAP CAM-As have intriguing properties in cell culture but also clear virus-infected cells from the mouse liver in a gradual and sustained way. We believe they represent a considerable improvement over previously reported molecules and may one day be part of curative treatment combinations for chronic hepatitis B.

Class A capsid assembly modulator RG7907 clears HBV-infected hepatocytes through core-dependent hepatocyte death and proliferation.

BACKGROUND AND AIMS: Effective therapies leading to a functional cure for chronic hepatitis B are still lacking. Class A capsid assembly modulators (CAM-As) are an attractive modality to address this unmet medical need. CAM-As induce aggregation of the HBV core protein (HBc) and lead to sustained HBsAg reductions in a chronic hepatitis B mouse model. Here, we investigate the underlying mechanism of action for CAM-A compound RG7907. APPROACH AND RESULTS: RG7907 induced extensive HBc aggregation in vitro , in hepatoma cells, and in primary hepatocytes. In the adeno-associated virus (AAV)-HBV mouse model, the RG7907 treatment led to a pronounced reduction in serum HBsAg and HBeAg, concomitant with clearance of HBsAg, HBc, and AAV-HBV episome from the liver. Transient increases in alanine transaminase, hepatocyte apoptosis, and proliferation markers were observed. These processes were confirmed by RNA sequencing, which also uncovered a role for interferon alpha and gamma signaling, including the interferon-stimulated gene 15 (ISG15) pathway. Finally, the in vitro observation of CAM-A-induced HBc-dependent cell death through apoptosis established the link of HBc aggregation to in vivo loss of infected hepatocytes. CONCLUSIONS: Our study unravels a previously unknown mechanism of action for CAM-As such as RG7907 in which HBc aggregation induces cell death, resulting in hepatocyte proliferation and loss of covalently closed circular DNA or its equivalent, possibly assisted by an induced innate immune response. This represents a promising approach to attain a functional cure for chronic hepatitis B.

Physiomimetic In Vitro Human Models for Viral Infection in the Liver.

Viral hepatitis is a leading cause of liver morbidity and mortality globally. The mechanisms underlying acute infection and clearance, versus the development of chronic infection, are poorly understood. In vitro models of viral hepatitis circumvent the high costs and ethical considerations of animal models, which also translate poorly to studying the human-specific hepatitis viruses. However, significant challenges are associated with modeling long-term infection in vitro. Differentiated hepatocytes are best able to sustain chronic viral hepatitis infection, but standard two-dimensional models are limited because they fail to mimic the architecture and cellular microenvironment of the liver, and cannot maintain a differentiated hepatocyte phenotype over extended periods. Alternatively, physiomimetic models facilitate important interactions between hepatocytes and their microenvironment by incorporating liver-specific environmental factors such as three-dimensional ECM interactions and co-culture with non-parenchymal cells. These physiologically relevant interactions help maintain a functional hepatocyte phenotype that is critical for sustaining viral hepatitis infection. In this review, we provide an overview of distinct, novel, and innovative in vitro liver models and discuss their functionality and relevance in modeling viral hepatitis. These platforms may provide novel insight into mechanisms that regulate viral clearance versus progression to chronic infections that can drive subsequent liver disease.

Roadblocks and fast tracks: How RNA binding proteins affect the viral RNA journey in the cell.

As obligate intracellular parasites with limited coding capacity, RNA viruses rely on host cells to complete their multiplication cycle. Viral RNAs (vRNAs) are central to infection. They carry all the necessary information for a virus to synthesize its proteins, replicate and spread and could also play essential non-coding roles. Regardless of its origin or tropism, vRNA has by definition evolved in the presence of host RNA Binding Proteins (RBPs), which resulted in intricate and complicated interactions with these factors. While on one hand some host RBPs recognize vRNA as non-self and mobilize host antiviral defenses, vRNA must also co-opt other host RBPs to promote viral infection. Focusing on pathogenic RNA viruses, we will review important scenarios of RBP-vRNA interactions during which host RBPs recognize, modify or degrade vRNAs. We will then focus on how vRNA hijacks the largest ribonucleoprotein complex (RNP) in the cell, the ribosome, to selectively promote the synthesis of its proteins. We will finally reflect on how novel technologies are helping in deepening our understanding of vRNA-host RBPs interactions, which can be ultimately leveraged to combat everlasting viral threats.

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

BACKGROUND & AIMS: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients. METHODS: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays. RESULTS: Transcriptomic analyses in cell culture and mouse models of coinfection enabled us to define an HDV-induced signature, mainly composed of interferon (IFN)-stimulated genes (ISGs). We also provide evidence that ISGs are upregulated in chronically HDV/HBV-coinfected patients but not in cells that only express HDV antigen (HDAg). Inhibition of the hepatocyte IFN response partially rescued the levels of HBV parameters. We observed less HBV RNA synthesis upon HDV infection or HDV protein expression. Additionally, HDV infection or expression of HDAg alone specifically accelerated the decay of HBV RNA, and HDAg was associated with HBV RNAs. On the contrary, HDAg expression did not affect other viruses such as HCV or SARS-CoV-2. CONCLUSIONS: Our data indicate that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms. Specifically, we uncover a new viral interference mechanism in which proteins of a satellite virus affect the RNA production of its helper virus. Exploiting these findings could pave the way to the development of new therapeutic strategies against HBV. IMPACT AND IMPLICATIONS: Although the molecular mechanisms remained unexplored, it has long been known that despite its dependency, HDV decreases HBV viremia in patients. Herein, using in vitro and in vivo models, we showed that HDV interferes with HBV through both IFN-dependent and IFN-independent mechanisms affecting HBV RNA metabolism, and we defined the HDV-induced modulation signature. The mechanisms we uncovered could pave the way for the development of new therapeutic strategies against HBV by mimicking and/or increasing the effect of HDAg on HBV RNA. Additionally, the HDV-induced modulation signature could potentially be correlated with responsiveness to IFN-α treatment, thereby helping to guide management of HBV/HDV-coinfected patients.

Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Scavenger receptor class B type I genetic variants associated with disease severity in chronic hepatitis C virus infection.

Analysis of host genetic polymorphisms is an increasingly important tool for understanding and predicting pathogenesis and treatment response of viral diseases. The gene locus of scavenger receptor class B type I (SR-BI), encoding a cell entry factor and receptor for hepatitis C virus (HCV), contains several genetic polymorphisms. We applied a probe extension assay to determine the frequency of six single nucleotide polymorphisms (SNPs) within the SR-BI gene locus in 374 individuals with history of HCV infection. In addition, SR-BI messenger RNA (mRNA) levels were analyzed in liver biopsy specimens of chronically infected HCV subjects. The rs5888 variant allele T was present at a higher frequency in subjects with advanced fibrosis (χ2 , p = 0.016) and after adjusting for age, duration of infection and alcohol intake as confounding factors. Haplotype analysis of SNP frequencies showed that a haplotype consisting of rs61932577 variant allele C and rs5888 variant allele T was associated with an increased risk of advanced liver fibrosis (defined by an Ishak score 4-6) (adjusted odds ratio 2.81; 95% confidence interval 1.06-7.46. p = 0.038). Carriers of the rs5888 variant allele T displayed reduced SR-BI mRNA expression in liver biopsy specimens. In conclusion the rs5888 polymorphism variant is associated with decreased SR-BI expression and an increased risk of development of advanced fibrosis in chronic HCV infection. These findings provide further evidence for a role of SR-BI in HCV pathogenesis and provides a genetic marker for prediction of those infected individuals at greater risk of developing severe disease.

Occludin stalls HCV particle dynamics apart from hepatocyte tight junctions, promoting virion internalization.

BACKGROUND AND AIMS: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated. APPROACH AND RESULTS: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization. CONCLUSIONS: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.

Observation of a Long-lived Electronic Coherence Modulated by Vibrational Dynamics in Molecular Nd3+ -Complexes at Room Temperature.

Temporally delayed, phase-locked coherent pairs of near IR femtosecond laser pulses were employed to study electronic coherences in molecular Nd3+ -complexes at room temperature. Dissolved and solid complexes were studied under a confocal microscope set-up with fluorescence detection. The observed electronic coherence on a few hundred femtoseconds time scale is modulated by additional coherent wave packet dynamics, which we attribute mainly to be vibrational in nature. In future, the complexes may serve as prototypes for possible applications in quantum information technology.

Cell-based cccDNA reporter assay combined with functional genomics identifies YBX1 as HBV cccDNA host factor and antiviral candidate target.

OBJECTIVES: Chronic hepatitis B virus (HBV) infection is a leading cause of liver disease and hepatocellular carcinoma. A key feature of HBV replication is the synthesis of the covalently close circular (ccc)DNA, not targeted by current treatments and whose elimination would be crucial for viral cure. To date, little is known about cccDNA formation. One major challenge to address this urgent question is the absence of robust models for the study of cccDNA biology. DESIGN: We established a cell-based HBV cccDNA reporter assay and performed a loss-of-function screen targeting 239 genes encoding the human DNA damage response machinery. RESULTS: Overcoming the limitations of current models, the reporter assay enables to quantity cccDNA levels using a robust ELISA as a readout. A loss-of-function screen identified 27 candidate cccDNA host factors, including Y box binding protein 1 (YBX1), a DNA binding protein regulating transcription and translation. Validation studies in authentic infection models revealed a robust decrease in HBV cccDNA levels following silencing, providing proof-of-concept for the importance of YBX1 in the early steps of the HBV life cycle. In patients, YBX1 expression robustly correlates with both HBV load and liver disease progression. CONCLUSION: Our cell-based reporter assay enables the discovery of HBV cccDNA host factors including YBX1 and is suitable for the characterisation of cccDNA-related host factors, antiviral targets and compounds.

Cystic fibrosis-related liver disease: Clinical presentations, diagnostic and monitoring approaches in the era of CFTR modulator therapies.

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population. Cystic fibrosis-related liver disease (CFLD) is defined as the pathogenesis related to the underlying CFTR defect in biliary epithelial cells. CFLD needs to be distinguished from other liver manifestations that may not have any pathological significance. The clinical/histological presentation and severity of CFLD vary. The main histological presentation of CFLD is focal biliary fibrosis, which is usually asymptomatic. Portal hypertension develops in a minority of cases (about 10%) and may require specific management including liver transplantation for end-stage liver disease. Portal hypertension is usually the result of the progression of focal biliary fibrosis to multilobular cirrhosis during childhood. Nevertheless, non-cirrhotic portal hypertension as a result of porto-sinusoidal vascular disease is now identified increasingly more frequently, mainly in young adults. To evaluate the effect of new CFTR modulator therapies on the liver, the spectrum of hepatobiliary involvement must first be precisely classified. This paper discusses the phenotypic features of CFLD, its underlying physiopathology and relevant diagnostic and follow-up approaches, with a special focus on imaging.

Liver Disease and Coronavirus Disease 2019: From Pathogenesis to Clinical Care.

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus that emerged in late 2019, is posing an unprecedented challenge to global health. Coronavirus disease 2019 (COVID-19), the clinical disease caused by SARS-CoV-2, has a variable presentation ranging from asymptomatic infection to life-threatening acute respiratory distress syndrome and multiorgan failure. Liver involvement is common during COVID-19 and exhibits a spectrum of clinical manifestations from asymptomatic elevations of liver function tests to hepatic decompensation. The presence of abnormal liver tests has been associated with a more severe presentation of COVID-19 disease and overall mortality. Although SARS-CoV-2 RNA has been detected in the liver of patients with COVID-19, it remains unclear whether SARS-CoV-2 productively infects and replicates in liver cells and has a direct liver-pathogenic effect. The cause of liver injury in COVID-19 can be attributed to multiple factors, including virus-induced systemic inflammation, hypoxia, hepatic congestion, and drug-induced liver disease. Among patients with cirrhosis, COVID-19 has been associated with hepatic decompensation and liver-related mortality. Additionally, COVID-19's impact on health care resources can adversely affect delivery of care and outcomes of patients with chronic liver disease. Understanding the underlying mechanisms of liver injury during COVID-19 will be important in the management of patients with COVID-19, especially those with advanced liver disease. This review summarizes our current knowledge of SARS-CoV-2 virus-host interactions in the liver as well the clinical impact of liver disease in COVID-19.