Measuring changes in cytosolic calcium levels in HBV- and HBx-expressing cultured primary hepatocytes

Abstract

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.