Hepatitis B Virus Infection of Hepatoma Cells Differentiated in Human Serum Culture
Creators: Le, Connie
Publisher: name: University of Alberta Library
Year: 2022

<p>Hepatitis B virus (HBV) has infected two billion people worldwide, culminating in approximately 250 million chronic carriers. Chronic HBV carriers are at high risk of developing severe liver diseases, such as cirrhosis and cancer, resulting in an estimated 600,000 HBV-associated deaths annually. A major obstacle to studies of HBV has been the lack of a biologically relevant and easily infectable cell culture model. Immortalized cell lines have low infection efficiency, while primary liver cells are difficult to acquire and maintain in culture. To overcome this problem, a human hepatoma cell culture system was developed for studying HBV infection. <br>
Overexpression of the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), in Huh7.5 hepatoma cells rendered them susceptible to HBV infection. The Huh7.5-NTCP hepatoma cells were differentiated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4% human serum (HS). This was compared to the conventional culture system using DMEM supplemented with fetal bovine serum (FBS) and requiring high concentrations (2–2.5%) of dimethyl sulfoxide (DMSO) to promote HBV infection. This new HS culture system produced robust HBV infection in Huh7.5-NTCP hepatoma cells in the absence of DMSO. HBV pregenomic RNA (pgRNA) levels in the HS cultures were increased by as much as 200-fold in comparison with FBS cultures and 19-fold in comparison with FBS+DMSO cultures. The HS culture increased levels of albumin secretion, a hepatocyte differentiation marker, in Huh7.5-NTCP cells to similar levels found in primary human hepatocytes. N-glycosylation of NTCP induced by HS-culture may contribute to viral entry. <br>
Single-cell RNA sequencing (scRNA-seq) analysis of Huh7.5-NTCP cells differentiated in HS cultures revealed that most of the cells had similar transcriptome profiles to those of primary hepatocytes from human liver. This transcriptomic characterization shows that the HS-cultured Huh7.5-NTCP cell model is a useful alternative to primary human hepatocytes. scRNA-seq analysis also revealed the presence of cholangiocyte-like cells in the HS-cultured Huh7.5 and Huh7.5-NTCP cell lines. The cholangiocyte-like cells in the hepatoma cell population had similar gene expression profiles and regulatory pathways to cholangiocytes from human liver tissue. <br>
scRNA-seq analyses were conducted to determine whether HBV infection alters interferon (IFN)-stimulated gene expression in Huh7.5 and Huh7.5-NTCP cells. Much higher amounts of the host cell RNA relative to low levels of HBV transcripts made the detection of HBV transcripts difficult. To overcome this problem, a CRISPR technique was developed to deplete highly abundant off-target transcripts and preferentially enrich the HBV transcripts. With CRISPR-mediated enrichment, scRNA-seq successfully detected HBV transcripts in more than 74% of the cells. This is compared to only 0.6% of the cells having detectable HBV transcripts when they were not enriched. The improved detection of HBV transcripts facilitated a scRNA-seq study of HBV infection and IFN treatment of Huh7.5-NTCP cells. Cells in the mock infection and HBV infection samples had the same transcriptome profiles. IFN treatment of Huh7.5-NTCP cells increased levels of IFN-stimulated genes. But HBV infection of the IFN-treated Huh7.5-NTCP cells did not alter the patterns or expression levels of IFN-stimulated genes. These results at the single-cell resolution support the idea that HBV is a “stealth virus”; it neither stimulates nor suppresses the interferon response. In contrast, hepatitis C virus (HCV) infection significantly changed transcriptome profiles of the host cells. HCV infection also altered expression patterns of IFN-stimulated genes, with upregulation of most and downregulation of a few IFN-stimulated genes.<br><br>
This study established an in vitro HBV infection model of Huh7.5-NTCP cells without using DMSO. scRNA-seq analysis of the cell model showed similar transcriptome profiles to those of primary human hepatocytes. scRNA-seq results also supported the hypothesis that HBV is a “stealth virus”. The discovery of cholangiocyte-like cells in HS-cultured Huh7.5-NTCP suggests a useful approach of cell differentiation. The tools and results described in this thesis contribute to an improved understanding of HBV infection.</p>