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

Many viruses modulate calcium (Ca 2+ ) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca 2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca 2+ 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 Ca 2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca 2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca 2+ signaling in cultured primary hepatocytes. We used single cell Ca 2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca 2+ level in hepatocytes following an IP 3 -linked Ca 2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca 2+ level required extracellular Ca 2+ influx and store-operated Ca 2+ (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 Ca 2+ 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 Ca 2+ 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 Ca 2+ accumulation to Ca 2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca 2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca 2+ signaling to regulate viral replication and virus-associated diseases.