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.