Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium, required for virus replication and cell death induction

Abstract

Infection with the hepatitis B virus (HBV) promotes the development of hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) and is a leading cause of morbidity and mortality worldwide. HBV X protein (HBx) is an important effector for HBV pathogenesis, but its cellular targets and acting mechanisms remain elusive. We show here that HBx interacts with the anti-apoptotic proteins Bcl-2 and Bcl-xL through a Bcl-2 homology 3 (BH3)-like motif in mammalian cells. Importantly, mutations in the BH3-like motif that prevent HBx binding to Bcl-2 and Bcl-xL abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication, which can be substantially rescued by restoring cytosolic calcium. These results suggest that HBx binding to Bcl-2 family members and subsequent elevation of cytosolic calcium are important for HBV viral replication. Consistently, RNAi knockdown of Bcl-2 or Bcl-xL results in reduced calcium elevation by HBx and decreased viral replication in hepatocytes. Our results suggest that HBx targets Bcl-2 proteins through its BH3-like motif to promote cytosolic calcium elevation, cell death, and viral replication during HBV pathogenesis, which presents an excellent therapeutic intervention point in treating patients with chronic HBV.calcium signaling | apoptosis | necrosis

Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx

“…This analysis is consistent with the observation that only high concentrations of HBx promoted apoptosis whereas low levels inhibited apoptosis (38). We cannot rule out the possibility that full-length HBx, which was used in the in vivo experiments (22,23), may have a higher binding affinity for Bcl-2 in cells. In addition, HBx has been reported to localize to mitochondria (39,40).…”

“…Previous studies suggest that the C-terminal BH3-like motif of HBx is crucial for apoptosis, cytosolic calcium increase, and HBV viral replication, whereas the antiapoptosis proteins Bcl-2 and Bcl-xL may represent the key cellular targets for such activities (22,23). Consistent with this hypothesis, C-terminal truncation of HBx is frequently detected in HCCs (35,36); compared with the full-length HBx, the truncated HBx (residues 1-127), which loses the C-terminal half of the BH3-like motif, could more effectively transform the liver cell line MIHA (36).…”

“…Experiments carried out in human hepatocytes suggest that Bcl-2 and Bcl-X L are the key cellular targets for HBx, where direct interactions between the BH3-like motif of HBx and the anti-apoptotic Bcl-2 family proteins trigger cytosolic calcium elevation, apoptosis, and viral DNA replication (22,23). The BH3-only proteins exemplified by Bim and Bad are thought to inhibit the anti-apoptosis proteins by binding to a conserved hydrophobic groove known as the BH3-binding groove (24)(25)(26).…”

Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca 2+ increase and cell death in Caenorhabditis elegans

“…CED-9, a key apoptosis regulator, was discovered unexpectedly to be a host target of HBx in cell death and Ca 2+ signaling in C. elegans, which led to identification of human Bcl-2 proteins as conserved host targets of HBx-mediated Ca 2+ stimulation and HBV replication in human hepatocytes (companion article, ref. 37). These findings demonstrate the validity of the C. elegans model for studying HBV and HBx.…”

“…Because Bcl-2 associates with HBx in HBV-infected hepatocytes (companion article, ref. 37), can substitute for CED-9 in mediating HBx-induced cell killing in C. elegans, and has been implicated in regulating MPT (39), Bcl-2 and Bcl-xL, both of which are mitochondrial proteins, are likely targeted by HBx during HBV infection to alter Ca 2+ signaling. The induced cytosolic Ca 2+ increase then triggers activation of multiple viral and host events, including HBV replication, assembly, and cell death.…”