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

Abstract

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.

Intrinsic hepatocyte dedifferentiation is accompanied by upregulation of mesenchymal markers, protein sialylation and core alpha 1,6 linked fucosylation

“…One pathway of interest was the AKT pathway, as the AKT1 gene was also up regulated in PRH as a function of time in culture22. Thus, in experiments similar to those described above, PRH were transfected with pMyrAkt1 or pMyrAkt2, which express myristolated forms of Akt 1 and 2 (see Methods), leading to constitutive Akt activation.…”

Hepatitis B virus molecular biology and pathogenesis

“…Although primary hepatocytes derived from small-animal models, namely rat and mouse, cannot be directly infected with HBV, they can support HBV replication and serve as a surrogate model system for studying the effects of HBV replication and HBV proteins on cellular physiology. [86, 185, 186] Here we provide a summary of the available cell culture model systems that are used to study HBV biology, each one possessing its own benefits and limitations.…”

“…[92] Recently, cultured primary rat hepatocytes have been used to study HBV replication and functions of the HBx protein; [83–86, 208] HBx activities in cultured primary rat hepatocytes were similar to HBx activities in cultured primary human hepatocytes, supporting the use of cultured primary rat hepatocytes as a model system for studying the impact of HBV on hepatocyte physiology. [86, 185, 186] …”

“…Recent RNA-seq analyses of HBV-expressing Huh7 cells [254] and primary rat hepatocytes [186] also detected decreased expression of GLUT2, the main hepatic glucose transporter. Investigation of the effect of fasting glucose levels on HBV replication revealed a link, albeit minor, between the metabolic state of the cell and the level of HBV replication, [245] and both gluconeogenesis and lipogenesis are under the same transcription factor control as HBV replication.…”

Clinical Trial Analysis of Different Stages of HBV Patients Treated with Human CIK Cells

“…The leading cause of primary hepatic cellular carcinoma is HBV infection, and it has been a serious problem worldwide [2,3]. The surrogate marker of hepatitis B virus DNA is hepatitis B e antigen (HBeAg).…”