The interplay of long noncoding RNAs and hepatitis B virus.

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.

Distinct Effects of Milk-Derived and Fermented Dairy Protein on Gut Microbiota and Cardiometabolic Markers in Diet-Induced Obese Mice.

BACKGROUND: Recent meta-analyses suggest that the consumption of fermented dairy products reduces type 2 diabetes and cardiovascular disease (CVD) risk, although the underlying mechanisms remain unclear. OBJECTIVE: We evaluated whether dairy protein products modulated gut microbiota and cardiometabolic features in mouse models of diet-induced obesity and CVD. METHODS: Eight-week-old C57BL/6J wild-type (WT) and LDLr-/-ApoB100/100 (LRKO) male mice were fed for 12 and 24 wk, respectively, with a high-fat/high-sucrose diet [66% kcal lipids, 22% kcal carbohydrates (100% sucrose), 12% kcal proteins]. The protein sources of the 4 diets were 100% nondairy protein (NDP), or 50% of the NDP energy replaced by milk (MP), milk fermented by Lactobacillus helveticus (FMP), or Greek-style yogurt (YP) protein. Fecal 16S rRNA gene-based amplicon sequencing, intestinal gene expression, and glucose tolerance test were conducted. Hepatic inflammation and circulating adhesion molecules were measured by multiplex assays. RESULTS: Feeding WT mice for 12 wk led to a 74% increase in body weight, whereas after 24 wk the LRKO mice had a 101.5% increase compared with initial body weight. Compared with NDP and MP, the consumption of FMP and YP modulated the gut microbiota composition in a similar clustering pattern, upregulating the Streptococcus genus in both genotypes. In WT mice, feeding YP compared with NDP increased the expression of genes involved in jejunal (Reg3b, 7.3-fold, P = 0.049) and ileal (Ocln, 1.7-fold, P = 0.047; Il1-ß,1.7-fold, P = 0.038; Nos2, 3.8-fold, P = 0.018) immunity and integrity. In LRKO mice, feeding YP compared with MP improved insulin sensitivity by 65% (P = 0.039). In LRKO mice, feeding with FMP versus NDP attenuated hepatic inflammation (monocyte chemoattractant protein 1, 2.1-fold, P ˂ 0.0001; IL1-ß, 5.7-fold, P = 0.0003; INF-γ, 1.7-fold, P = 0.002) whereas both FMP [vascular adhesion molecule 1 (VCAM1), 1.3-fold, P = 0.0003] and YP (VCAM1, 1.04-fold, P = 0.013; intracellular adhesion molecule 1, 1.4-fold, P = 0.028) decreased circulating adhesion molecules. CONCLUSION: Both fermented dairy protein products reduce cardiometabolic risk factors in diet-induced obese mice, possibly by modulating the gut microbiota.

Transcriptome-Wide Analysis of Hepatitis B Virus-Mediated Changes to Normal Hepatocyte Gene Expression.

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.

Countywide implementation of crisis intervention teams: Multiple methods, measures and sustained outcomes.

The crisis intervention team (CIT) is a tool that can be used to foster pre-booking diversion of individuals with mental illness from the criminal justice system and into community treatment services. Although CIT is often implemented solely as the training of law enforcement officers, the model stipulates that CIT is a vehicle for collaboration with community stakeholders who share a similar philosophy, as well as expanded mental health services offering a 24 hour-seven days per week drop-off option for law enforcement officers. This case study presents the countywide implementation of CIT and expands previous findings on the prevalence of officer interaction with persons with mental health issues and CIT training outcomes, including changes in officer perception of individuals with mental health issues. Furthermore, analysis of the disposition of calls for officer assistance coded as mental health or suicide found significant increases in officer drop-offs to the mental health crisis center post-CIT training. Interrupted time series analysis determined that this change has been sustained over time, perhaps owing to the unique communication between county law enforcement and mental health staff. Implications for policy and practice are discussed.

The hepatitis B virus (HBV) HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival.

UNLABELLED: Chronic infection with hepatitis B virus (HBV) is a risk factor for developing liver diseases such as hepatocellular carcinoma (HCC). HBx is a multifunctional protein encoded by the HBV genome; HBx stimulates HBV replication and is thought to play an important role in the development of HBV-associated HCC. HBx can activate the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway in some cell lines; however, whether HBx regulates PI3K/AKT signaling in normal hepatocytes has not been evaluated. In studies described here, we assessed HBx activation of PI3K/AKT signaling in an ex vivo model of cultured primary hepatocytes and determined how this HBx activity affects HBV replication. We report that HBx activates AKT in primary hepatocytes and that the activation of AKT decreases HBV replication and HBV mRNA and core protein levels. We show that the transcription factor hepatocyte nuclear factor 4α (HNF4α) is a target of HBx-regulated AKT, and we link HNF4α to HBx-regulated AKT modulation of HBV transcription and replication. Although we and others have shown that HBx stimulates and is likely required for HBV replication, we now report that HBx also activates signals that can diminish the overall level of HBV replication. While this may seem counterintuitive, we show that an important effect of HBx activation of AKT is inhibition of apoptosis. Consequently, our studies suggest that HBx balances HBV replication and cell survival by stimulating signaling pathways that enhance hepatocyte survival at the expense of higher levels of HBV replication. IMPORTANCE: Chronic hepatitis B virus (HBV) infection is a common cause of the development of liver cancer. Regulation of cell signaling pathways by the HBV HBx protein is thought to influence the development of HBV-associated liver cancer. HBx stimulates, and may be essential for, HBV replication. We show that HBx activates AKT in hepatocytes to reduce HBV replication. While this seems contradictory to an essential role of HBx during HBV replication, HBx activation of AKT inhibits hepatocyte apoptosis, and this may facilitate persistent, noncytopathic HBV replication. AKT regulates HBV replication by reducing the activity of the transcription factor hepatocyte nuclear factor 4α (HNF4α). HBx activation of AKT may contribute to the development of liver cancer by facilitating persistent HBV replication, augmenting the dedifferentiation of hepatocytes by inhibiting HNF4α functions, and activating AKT-regulated oncogenic pathways. AKT-regulated factors may provide therapeutic targets for inhibiting HBV replication and the development of HBV-associated liver cancer.

Technical standards for hepatitis B virus X protein (HBx) research.

Chronic infection with hepatitis B virus (HBV) is a risk factor for developing hepatocellular carcinoma (HCC). The life cycle of HBV is complex and has been difficult to study because HBV does not infect cultured cells. The HBV regulatory X protein (HBx) controls the level of HBV replication and possesses an HCC cofactor role. Attempts to understand the mechanism(s) that underlie HBx effects on HBV replication and HBV-associated carcinogenesis have led to many reported HBx activities that are likely influenced by the assays used. This review summarizes experimental systems commonly used to study HBx functions, describes limitations of these experimental systems that should be considered, and suggests approaches for ensuring the biological relevance of HBx studies.

Protein phosphatase 2A (PP2A) regulates low density lipoprotein uptake through regulating sterol response element-binding protein-2 (SREBP-2) DNA binding.

LDL-cholesterol (LDL-C) uptake by Ldlr is regulated at the transcriptional level by the cleavage-dependent activation of membrane-associated sterol response element-binding protein (SREBP-2). Activated SREBP-2 translocates to the nucleus, where it binds to an LDLR promoter sterol response element (SRE), increasing LDLR gene expression and LDL-C uptake. SREBP-2 cleavage and translocation steps are well established. Several SREBP-2 phosphorylation sites have been mapped and functionally characterized. The phosphatases dephosphorylating these sites remain elusive. The phosphatase(s) regulating SREBP-2 represents a novel pharmacological target for treating hypercholesterolemia. Here we show that protein phosphatase 2A (PP2A) promotes SREBP-2 LDLR promoter binding in response to cholesterol depletion. No binding to an LDLR SRE was observed in the presence of the HMG-CoA reductase inhibitor, lovastatin, when PP2A activity was inhibited by okadaic acid or depleted by siRNA methods. SREBP-2 cleavage and nuclear translocation were not affected by loss of PP2A. PP2A activity was required for SREBP-2 DNA binding. In response to cholesterol depletion, PP2A directly interacted with SREBP-2 and altered its phosphorylation state, causing an increase in SREBP-2 binding to an LDLR SRE site. Increased binding resulted in induced LDLR gene expression and increased LDL uptake. We conclude that PP2A activity regulates cholesterol homeostasis and LDL-C uptake.

Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms.

We describe the generation of microfluidic platforms for the co-culture of primary hepatocytes and endothelial cells; these platforms mimic the architecture of a liver sinusoid. This paper describes a progressional study of creating such a liver sinusoid on a chip system. Primary rat hepatocytes (PRHs) were co-cultured with primary or established endothelial cells in layers in single and dual microchannel configurations with or without continuous perfusion. Cell viability and maintenance of hepatocyte functions were monitored and compared for diverse experimental conditions. When primary rat hepatocytes were co-cultured with immortalized bovine aortic endothelial cells (BAECs) in a dual microchannel with continuous perfusion, hepatocytes maintained their normal morphology and continued to produce urea for at least 30 days. In order to demonstrate the utility of our microfluidic liver sinusoid platform, we also performed an analysis of viral replication for the hepatotropic hepatitis B virus (HBV). HBV replication, as measured by the presence of cell-secreted HBV DNA, was successfully detected. We believe that our liver model closely mimics the in vivo liver sinusoid and supports long-term primary liver cell culture. This liver model could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, cancer research, and analysis of pathological effects and replication strategies of various hepatotropic infectious agents. .

The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake.

Chronic hepatitis B virus (HBV) infections are associated with the development of hepatocellular carcinoma (HCC). The HBV X protein (HBx) is thought to play an important role in the development of HBV-associated HCC. One fundamental HBx function is elevation of cytosolic calcium signals; this HBx activity has been linked to HBx stimulation of cell proliferation and transcription pathways, as well as HBV replication. Exactly how HBx elevates cytosolic calcium signals is not clear. The studies described here show that HBx stimulates calcium entry into cells, resulting in an increased plateau level of inositol 1,4,5-triphosphate (IP3)-linked calcium signals. This increased calcium plateau can be inhibited by blocking mitochondrial calcium uptake and store-operated calcium entry (SOCE). Blocking SOCE also reduced HBV replication. Finally, these studies also demonstrate that there is increased mitochondrial calcium uptake in HBx-expressing cells. Cumulatively, these studies suggest that HBx can increase mitochondrial calcium uptake and promote increased SOCE to sustain higher cytosolic calcium and stimulate HBV replication.

Hypoxia-inducible factor affects hepatitis B virus transcripts and genome levels as well as the expression and subcellular location of the hepatitis B virus core protein.

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.

The hepatitis B virus X protein modulates hepatocyte proliferation pathways to stimulate viral replication.

Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G(1) phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G(1) phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.

Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore.

The hepatitis B virus (HBV) X protein (HBx) is a multifunctional protein that regulates numerous cellular signal transduction pathways, including those that modulate apoptosis. However, different HBx-dependent effects on apoptosis have been reported; these differences are likely the consequence of the exact conditions and cell types used in a study. Many of the previously reported studies that analyzed HBx regulation of apoptosis were conducted in immortalized or transformed cells, and the alterations that have transformed or immortalized these cells likely impact apoptotic pathways. We examined the effects of HBx on apoptotic pathways in cultured primary rat hepatocytes, a biologically relevant system that mimics normal hepatocytes in the liver. We analyzed the effects of HBx on apoptosis both when HBx was expressed in the absence of other HBV proteins and in the context of HBV replication. HBx stimulation of NF-kappaB inhibited the activation of apoptotic pathways in cultured primary rat hepatocytes. However, when HBx-induced activation of NF-kappaB was blocked, HBx stimulated apoptosis; blocking the activity of the mitochondrial permeability transition pore inhibited HBx activation of apoptosis. These results suggest that HBx can be either proapoptotic or antiapoptotic in hepatocytes, depending on the status of NF-kappaB, and confirm previous studies that link some HBx activities to modulation of the mitochondrial permeability transition pore. Overall, our studies define apoptotic pathways that are regulated by HBx in cultured primary hepatocytes and provide potential mechanisms for the development of HBV-associated liver cancer.

Dietary sucrose induces metabolic inflammation and atherosclerotic cardiovascular diseases more than dietary fat in LDLr-/-ApoB100/100 mice.

BACKGROUND AND AIMS: Poor dietary habits contribute to the obesity pandemic and related cardiovascular diseases but the respective impact of high saturated fat versus added sugar consumption remains debated. Herein, we aimed to disentangle the individual role of dietary fat versus sugar in cardiometabolic disease progression. METHODS: We fed pro-atherogenic LDLr-/-ApoB100/100 mice either a low-fat/high-sucrose (LFHS) or a high-fat/low-sucrose (HFLS) diet for 24 weeks. Weekly body weight gain was registered. 16S rRNA gene-based gut microbial analysis was performed to investigate gut microbial modulations. Intraperitoneal insulin (ipITT) and oral glucose tolerance test (oGTT) were conducted to assess glucose homeostasis and insulin sensitivity. Cytokines were assessed in fasted plasma, epididymal white adipose tissue and liver lysates. Heart function was evaluated by echocardiography. Aortic atheroma lesions were quantified according to the en face technique. RESULTS: HFLS feeding increased obesity, insulin resistance and dyslipidemia compared to LFHS feeding. Conversely, high sucrose consumption decreased gut microbial diversity while augmenting inflammation and the adaptative immune defense against metabolic endotoxemia and reduced macrophage cholesterol efflux capacity. This led to more severe cardiovascular complications as revealed by remarkably high level of atherosclerotic lesions and the early development of cardiac dysfunction in LFHS vs HFLS fed mice. CONCLUSIONS: We uncoupled obesity-associated insulin resistance from cardiovascular diseases and provided novel evidence that dietary sucrose, not fat, is the main driver of metabolic inflammation accelerating severe atherosclerosis in hyperlipidemic mice.

Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential.

Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-kappaB activity. In primary rat hepatocytes, HBx activation of NF-kappaB prevented mitochondrial membrane depolarization; however, when NF-kappaB activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.

Hepatitis B virus X protein modulates cytosolic Ca2+ signaling in primary human hepatocytes.

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.

Role of HBx in hepatitis B virus persistence and its therapeutic implications.

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.

A broad investigation of the HBV-mediated changes to primary hepatocyte physiology reveals HBV significantly alters metabolic pathways.

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.

Measuring Changes in Cytosolic Calcium Levels in HBV- and HBx-Expressing Cultured Primary Hepatocytes.

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.

Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

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.

Hepatitis B Virus X and Regulation of Viral Gene Expression.

The efficient replication of hepatitis B virus (HBV) requires the HBV regulatory hepatitis B virus X (HBx) protein. The exact contributions of HBx are not fully understood, in part because of the limitations of the assays used for its study. When HBV replication is driven from a plasmid DNA, the contribution of HBx is modest. However, there is an absolute requirement for HBx in assays that recapitulate the infectious virus life cycle. There is much evidence that HBx can contribute directly to HBV replication by acting on viral promoters embedded within protein coding sequences. In addition, HBx may also contribute indirectly by modulating cellular pathways to benefit virus replication. Understanding the mechanism(s) of HBx action during virus replication may provide insight into novel ways to disrupt chronic HBV replication.