Abstract
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.
Highlights
Viruses are obligate intracellular parasites, and many viruses have developed methods to subvert host cell signal transduction pathways and factors to support their own replication and survival [1]
We previously demonstrated that cultured primary rat hepatocytes, which are readily available, can serve as a surrogate model for studying hepatitis B virus (HBV) effects in primary human hepatocytes, which are not always available in large quantities
Reverse transcription-quantitative PCR (RT-qPCR) was performed to quantitate levels of the hepatocyte-specific markers albumin, transferrin, and hepatocyte nuclear factor 4 alpha (HNF4α) [38,39,40]. These markers were expressed at high levels in hepatocytes immediately following isolation and in hepatocytes that were maintained in culture for 48 hours, the longest time frame for our experiments (Fig 1A)
Summary
Viruses are obligate intracellular parasites, and many viruses have developed methods to subvert host cell signal transduction pathways and factors to support their own replication and survival [1]. The amplitude, frequency, and spatiotemporal patterning of an intracellular Ca2+ signal can elicit differential modulation of Ca2+-binding proteins and Ca2+-dependent effectors, thereby regulating numerous cellular responses and functions [5, 6]. Many pathogens, including a large number of DNA and RNA viruses, encode proteins that alter normal cellular Ca2+ signaling; for viruses, altered Ca2+ signaling typically enhances viral replication [6, 7]. Due to the versatile nature of a Ca2+ signal, modulation of intracellular Ca2+. Signaling is an ideal mechanism for viruses to create a cellular environment that is permissive to viral replication.
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