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.
Program in Molecular and Cellular Biology and Genetics, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
Jessica C
JC
Casciano
Program in Molecular and Cellular Biology and Genetics, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
Nicholas J
NJ
Duchemin
Program in Microbiology and Immunology, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
R Jason
RJ
Lamontagne
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
Laura F
LF
Steel
Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.
Michael J
MJ
Bouchard
D000818:Animals; D020013:Calcium Signaling / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D006515:Hepatitis B virus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000378:metabolism / Q000821:virology; D022781:Hepatocytes / Q000378:metabolism* / Q000821:virology*; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000502:physiology; D006801:Humans; D051381:Rats; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D014764:Viral Proteins / Q000502:physiology; D054334:Viral Regulatory and Accessory Proteins; D014779:Virus Replication / Q000235:genetics / Q000502:physiology*
D015534:Trans-Activators; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein