Primary liver cancer represents a significant global health burden, with limited therapeutic options for advanced disease. Long non-coding RNAs (lncRNAs) are increasingly found to play crucial roles in hepatic biology and disease progression. Here, we identify FAM99A as a highly liver-enriched lncRNA that is systematically downregulated across liver malignancies, with reduced expression correlating with poor clinical outcomes. FAM99A exhibits remarkable tissue specificity with minimal expression outside the liver, and its levels rapidly decline during primary hepatocyte dedifferentiation in culture. Through isoform analysis, we establish FAM99A-203 as the predominant transcript in normal liver tissue and observe altered isoform distribution in liver cancers. Functionally, FAM99A overexpression inhibits anchorage-independent growth in liver cancer cell lines. Transcriptomic analysis reveals that FAM99A negatively regulates translation-related pathways in both liver cancer cells and primary hepatocytes. This is corroborated by protein synthesis assays showing that FAM99A overexpression substantially reduces global translation rates. Targeted RNase H-mediated extraction coupled with mass spectrometry identifies multiple components of the translation machinery as direct FAM99A binding partners, including eukaryotic translation initiation factors and RNA helicases involved in ribosome biogenesis. Clinical data analysis demonstrates significant inverse correlations between FAM99A expression and ribosomal protein genes in liver cancer patients. Additionally, hepatitis B virus appears to downregulate FAM99A expression, potentially contributing to its oncogenic properties. Our findings establish FAM99A as a liver-enriched translational regulator that exerts tumor-suppressive effects by restraining protein synthesis rates, offering insights into hepatocarcinogenesis and the potential of FAM99A as both a biomarker and agent in new therapeutic avenues.

Globally, a chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The increased risk of HCC persists in patients treated with antiviral therapy, despite suppressing viral replication. Co-infection with human immunodeficiency virus (HIV) increases the risk of developing HCC by 18-fold relative to an HBV mono-infection. Despite the clear clinical burden posed by an HIV/HBV co-infection, the mechanisms underlying the increased risk of HCC are understudied due to a lack of relevant model systems. We developed an in vitro model of HIV/HBV co-infection using Jurkat cells with inducible expression of HIV-1 Tat co-cultured with HBV-infected primary rat hepatocytes. The co-culture model was used to investigate the effect of HIV-1 Tat on HBV replication and cell survival in HBV-infected hepatocytes. Results from Jurkat-secreted Tat were complemented with purified HIV-1 Tat to determine the effect of Tat alone. Exposure to Tat significantly upregulated ERK1/2 and NF-[kappa]B signaling and cell survival in HBV-infected and uninfected hepatocytes. The activation of ERK1/2 and NF-[kappa]B signaling by Tat also inhibited HBV replication, which could be rescued by inhibition of ERK1/2 with temuterkib. Tat-dependent activation of NF-[kappa]B signaling and cell survival was also reversed by treatment with temuterkib. Tat-mediated activation of NF-[kappa]B signaling and cell survival is initiated by the TLR-4 cell surface receptor and recruitment of PKC-delta to the plasma membrane. Overall, the results of these studies indicate that Tat-mediated upregulation of ERK1/2 may promote oncogenesis and contribute to the development of HCC during an HIV/HBV co-infection by promoting cell survival.

Liver macrophages play important roles in the pathophysiology of liver fibrosis and hepatocellular carcinoma. However, liver macrophages are a heterogenous population and have differing roles in maintenance of liver function and response in disease. In a healthy liver, macrophages play a critical role in antigen processing, maintaining tolerance to the high levels of gut-derived bacterial products, and regulating inflammation through cytokine response. However, macrophages also play a critical role in liver pathology, specifically in the context of viral infection. The liver is targeted by multiple viruses, including human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, which dysregulate macrophage functions to affect liver disease. Infection with any of these viruses is associated with increased risk of developing hepatocellular carcinoma, and coinfection further accelerates the progression to liver disease and cancer. However, the exact mechanisms by which liver macrophages contribute to disease in the context of viral infections are not well defined. This is a particularly acute issue in human immunodeficiency virus-infected populations, which have high incidence of hepatitis B virus and hepatitis C virus coinfection. To address this knowledge gap, this review describes the populations of macrophages in the liver, outlines the current models and limitations associated with the study of liver macrophages, discusses the function and role of liver macrophages in the context of viral infection, and describes the mechanisms by which these cells contribute to hepatocellular carcinoma and fibrosis. We then use this information to propose focus areas for the liver macrophage field to better address and resolve viral liver disease.

Infection by hepatitis B virus (HBV) and hepatitis D virus (HDV) is a major health threat worldwide. In 2019 approximately 296 million individuals were living with a chronic HBV infection. Globally, a chronic HBV infection is the most common risk factor for developing hepatocellular carcinoma (HCC), a leading cause of cancer-related mortality. HDV is a satellite virus of HBV and requires the HBV surface protein for genome packaging and transmission. Individuals with pre-existing chronic HBV infection who contract HDV, resulting in an HDV superinfection, have an increased risk of developing a chronic HBV/HDV infection, which greatly increases the risk of developing HCC compared to a chronic HBV infection. This chapter will describe the molecular biology, clinical features and pathogenesis, epidemiology, and methods to detect HBV and HDV. Infection-prevention strategies, current therapies, and mechanisms that could link a chronic HBV or HBV/HDV infection to HCC development will also be discussed.

Globally, a chronic-hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC). The transcription factor hypoxia-inducible factor 1 (HIF1) is often elevated in HCC, including HBV-associated HCC. Previous studies have suggested that the expression of the HIF1 subunit, HIF1α, is elevated in HBV-infected hepatocytes; however, whether HIF1 activity affects the HBV lifecycle has not been fully explored. We used a liver-derived cell line and ex vivo cultured primary hepatocytes as models to determine how HIF1 affects the HBV lifecycle. We observed that HIF1 elevates HBV RNA transcript levels, core protein levels, core protein localization to the cytoplasm, and HBV genome replication. Attenuating the transcription activity of HIF1 blocked HIF1-mediated effects on the HBV lifecycle. Our studies show that HIF1 regulates various stages of the HBV lifecycle in hepatocytes and could be a therapeutic target for blocking HBV replication and the development of HBV-associated diseases.

Hepatitis B Virus (HBV) infections remain a major global health burden with an estimated 296 million people living with a chronic infection and 884,000 HBV-related deaths annually. Notably, patients with a chronic hepatitis B (CHB) infection are at a 30-fold greater risk of developing hepatocellular carcinoma (HCC), the most common type of primary liver cancer, which is the 3rd deadliest cancer worldwide. Several groups have assessed HBV-related aberrant expression of host-cell long noncoding RNAs (lncRNAs) and how altered expression of specific lncRNAs affects HBV replication and progression to associated disease states. Given the challenges in establishing effective HBV models and analyzing transcriptomic data, this review focuses on lncRNA expression data primarily collected from clinical patient samples and primary human hepatocytes, with the subsequent mechanism of action analysis in cell lines or other model systems. Ultimately, understanding HBV-induced lncRNA-expression dysregulation could lead to new treatments and biomarkers for HBV infection and its associated diseases.

Worldwide, approximately 240 million people are chronically infected with the hepatitis B virus (HBV); chronic HBV infection is associated with the development of life-threatening liver diseases. The HBV HBx protein alters hepatocyte physiology to promote HBV replication. We previously reported that HBx modulates calcium signaling to stimulate HBV replication in human hepatoblastoma HepG2 cells and primary rat hepatocytes. Whether HBx modulates calcium signaling in a primary human hepatocyte, the natural site of an HBV infection, has not been determined. Here, we report the effect of HBx on calcium signaling in primary human hepatocytes and show that HBx modulates calcium signaling via enhanced calcium entry through store-operated calcium channels and elevated mitochondrial calcium, similar to HBx effects in HepG2 cells and primary rat hepatocytes. In addition to demonstrating that HBV and HBx affect calcium signaling in human hepatocytes, these studies also show that HBV and HBx regulation of calcium signaling is identical in primary human and rat hepatocytes, further validating the use of cultured primary rat hepatocytes for HBV studies.

Objective: As the leading risk factor for the development of liver cancer, chronic infection with hepatitis B virus (HBV) represents a significant global health concern. Although an effective HBV vaccine exists, at least 240 million people are chronically infected with HBV worldwide. Therapeutic options for the treatment of chronic HBV remain limited, and none achieve an absolute cure. To develop novel therapeutic targets, a better understanding of the complex network of virus-host interactions is needed. Because of the central metabolic role of the liver, we assessed the metabolic impact of HBV infection as a means to identify viral dependency factors and metabolic pathways that could serve as novel points of therapeutic intervention. Methods: Primary rat hepatocytes were infected with a control adenovirus, an adenovirus expressing a greater than-unit-length copy of the HBV genome, or an adenovirus expressing the HBV X protein (HBx). A panel of 369 metabolites was analyzed for HBV- or HBx-induced changes 24 and 48 h post infection. Pathway analysis was used to identify key metabolic pathways altered in the presence of HBV or HBx expression, and these findings were further supported through integration of publically available gene expression data. Results: We observed distinct changes to multiple metabolites in the context of HBV replication or HBx expression. Interestingly, a panel of 7 metabolites (maltotriose, maltose, myristate [14:0], arachidate [20:0], 3-hydroxybutyrate [BHBA], myo-inositol, and 2-palmitoylglycerol [16,0]) were altered by both HBV and HBx at both time points. In addition, incorporation of data from a transcriptome-based dataset allowed us to identify metabolic pathways, including long chain fatty acid metabolism, glycolysis, and glycogen metabolism, that were significantly altered by HBV and HBx. Conclusions: Because the liver is a central regulator of metabolic processes, it is important to understand how HBV replication and HBV protein expression affects the metabolic function of hepatocytes. Through analysis of a broad panel of metabolites we investigated this metabolic impact. The results of these studies have defined metabolic consequences of an HBV infection of hepatocytes and will help to lay the groundwork for novel research directions and, potentially, development of novel anti-HBV therapeutics. (C) 2018 Elsevier Inc. All rights reserved.

A chronic hepatitis B virus (HBV) infection can cause liver cancer, specifically hepatocellular carcinoma (HCC). A variety of mechanisms that can lead from HBV infection to development of HCC have been proposed and could include prolonged expression of HBV proteins that alter cellular signaling pathways, recurrent anti-HBV inflammatory responses associated with death of HBV-infected hepatocytes and concomitant liver regeneration, and integration of the HBV genome into the host cell chromosome, which could alter expression of cellular genes. Because it typically requires decades for a chronic HBV infection to cause liver cancer, it has been difficult to identify a single HBV-related effect that is carcinogenic, and it is likely that subtle HBV-associated effects over a prolonged period of time cause HCC. Anti-HBV therapies, such as interferon or nucleoside analogs can successfully control HBV replication and delay development of diseases associated with an HBV infection; however, these treatments are typically not curative. In this chapter, mechanisms that have been proposed to contribute to the progression and development of HBV-associated HCC, as well as established and emerging anti-HBV therapeutic strategies, will be discussed.

Chronic hepatitis B virus infection is a significant risk factor for cirrhosis and hepatocellular carcinoma. The HBx protein is required for virus replication, but the lack of robust infection models has hindered our understanding of HBx functions that could be targeted for antiviral purposes. We briefly review three properties of HBx: its binding to DDB1 and its regulation of cell survival and metabolism, to illustrate how a single viral protein can have multiple effects in a cell. We propose that different functions of HBx are needed, depending on the changing hepatocyte environment encountered during a chronic virus infection, and that these functions might serve as novel therapeutic targets for inhibiting hepatitis B virus replication and the development of associated diseases.

Chronic infection with hepatitis B virus (HBV) remains a major worldwide health concern and is the leading cause of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is the only regulatory protein encoded in the HBV genome; HBx stimulates HBV replication in vivo and in vitro. HBx also regulates cytosolic Ca2+ signaling, and altered Ca2+ signaling is associated with the development of many diseases, including HCC. Importantly, many HBx functions, including HBx modulation of cell proliferation, apoptosis, and transcription pathways, have been linked to changes in cytosolic Ca2+ signaling. Additionally, several stages of HBV replication, including capsid formation and activation of the HBV polymerase, are dependent on intracellular Ca2+. Consequently, defining the molecular mechanism that underlies HBV and HBx modulation of cytosolic Ca2+ levels is important for understanding HBV pathogenesis and the role of HBx in HBV replication. Here, we describe a single-cell Ca2+-imaging protocol that we use to investigate HBV and HBx effects on the level of cytosolic Ca2+. We specifically outline two methods that we use to evaluate HBV and HBx regulation of cytosolic Ca2+ levels in cultured primary hepatocytes. This protocol can also be adapted for use in liver cell lines.

Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome- wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

We have developed a miniature human liver (liver-sinusoid-on-a-chip) model using a dual microchannel separated by a porous membrane. Primary human hepatocytes and immortalized bovine aortic endothelial cells were co-cultured on opposite sides of a microporous membrane in a dual microchannel with continuous perfusion. Primary human hepatocytes in this system retained their polygonal morphology for up to 26 days, while hepatocytes cultured in the absence of bovine aortic endothelial cells lost their morphology within a week. In order to demonstrate the utility of our human-liver-sinusoid-on-a-chip, human hepatocytes in this system were directly infected by Hepatitis B Virus (HBV). Expression of the HBV core antigen was detected in human hepatocytes in the microchannel system. HBV replication, measured by the presence of cell-secreted HBV DNA, was also detected. Importantly, HBV is hepatotropic, and expression of HBV RNA transcripts is dependent upon expression of hepatocyte-specific factors. Moreover, HBV infection requires expression of the human-hepatocyte-specific HBV cell surface receptor. Therefore, the ability to detect HBV replication and Hepatitis B core Antigen (HBcAg) expression in our microfluidic platform confirmed that hepatocyte differentiation and functions were retained throughout the time course of our studies. We believe that our human-liver-sinusoid-on-a-chip could have many applications in liver-related research and drug development.

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350-500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae. In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC.

Alterations in N-linked glycosylation have long been associated with cancer but for the most part, the reasons why have remained poorly understood. Here we show that increased core fucosylation is associated with de-differentiation of primary hepatocytes and with the appearance of markers indicative of a transition of cells from an epithelial to a mesenchymal state. This increase in core fucosylation was associated with increased levels of two enzymes involved in α-1,6 linked fucosylation, GDP-mannose 4, 6-dehydratase (Gmds) and to a lesser extent fucosyltransferase 8 (Fut8). In addition, the activation of cancer-associated cellular signaling pathways in primary rat hepatocytes can increase core fucosylation and induce additional glycoform alterations on hepatocyte proteins. Specifically, we show that increased levels of protein sialylation and α-1,6-linked core fucosylation are observed following activation of the β-catenin pathway. Activation of the Akt signaling pathway or induction of hypoxia also results in increased levels of fucosylation and sialylation. We believe that this knowledge will help in the better understanding of the genetic factors associated with altered glycosylation and may allow for the development of more clinically relevant biomarkers.

Globally, a chronic hepatitis B virus (HBV) infection remains the leading cause of primary liver cancer. The mechanisms leading to the development of HBV-associated liver cancer remain incompletely understood. In part, this is because studies have been limited by the lack of effective model systems that are both readily available and mimic the cellular environment of a normal hepatocyte. Additionally, many studies have focused on single, specific factors or pathways that may be affected by HBV, without addressing cell physiology as a whole. Here, we apply RNA-seq technology to investigate transcriptome-wide, HBV-mediated changes in gene expression to identify single factors and pathways as well as networks of genes and pathways that are affected in the context of HBV replication. Importantly, these studies were conducted in an ex vivo model of cultured primary hepatocytes, allowing for the transcriptomic characterization of this model system and an investigation of early HBV-mediated effects in a biologically relevant context. We analyzed differential gene expression within the context of time-mediated gene-expression changes and show that in the context of HBV replication a number of genes and cellular pathways are altered, including those associated with metabolism, cell cycle regulation, and lipid biosynthesis. Multiple analysis pipelines, as well as qRT-PCR and an independent, replicate RNA-seq analysis, were used to identify and confirm differentially expressed genes. HBV-mediated alterations to the transcriptome that we identified likely represent early changes to hepatocytes following an HBV infection, suggesting potential targets for early therapeutic intervention. Overall, these studies have produced a valuable resource that can be used to expand our understanding of the complex network of host-virus interactions and the impact of HBV-mediated changes to normal hepatocyte physiology on viral replication.