HEPATITIS B VIRUS
C. elegans as a new animal model for treating HBV
Hepatitis B Virus (HBV) is the smallest human DNA virus and the major cause of acute and chronic liver disease including hepatocellular carcinoma (HCC) worldwide. There are ~ 400 million HBV chronic carriers and 600,000 deaths every year. Little is known about how HBV infection causes liver injuries and induces the development of liver cancer, partly due to the lack of an effective animal model amenable to genetic analysis. Neither is there an effective treatment for this disease. HBV infection causes liver injuries by inducing liver cell necrosis and apoptosis, which could cause cycles of inflammatory cytokine release, local liver damage, and compensatory regeneration, leading to the continual acquisition of oncogenic mutations and the development of HCC. We tested the possibility of using C. elegans as an animal model to study the mechanisms of HBV-induced cell degeneration. We found that expression of the HBV X protein (HBx) in C.elegans induces mostly necrosis and some apoptosis, mimicking the early event of liver infection by HBV. The mechanisms of action of HBx are unknown, but in vitro it can activate multiple signaling pathways and interact with many different proteins. Transgenic mice expressing HBx in the liver display widespread liver cell deaths and later develop liver cirrhosis and HCC. To identify in vivo targets and signaling pathways of HBx, we carried out genetic screens to isolate suppressors of HBx-induced cell death and obtained 31 mutations, which define at least ten genes (hids-1 to hids-10; HBx-induced death suppressors).
In parallel to the genetic suppressor screens, we performed genetic and biochemical analyses of HBx-induced cell death in C. elegans and found unexpectedly thatHBx interacts directly with CED-9, a human Bcl-2 homolog, through a Bcl-2 homology 3 (BH3)-like motif and this interaction triggers both cytosolic Ca2+increase and cell death. Similarly, two mammalian Bcl-2 family proteins, Bcl-2 and Bcl-xL, also interact with HBx through the same BH3 motif in human hepatocytes. Importantly, mutations in the BH3-like motif that prevent HBx binding to the Bcl-2 proteins abrogate cytosolic calcium elevation and cell death induced by HBx expression in hepatocytes and severely impair HBV viral replication. RNAi knockdown of Bcl-2 or Bcl-xL also results in decreased viral replication in hepatocytes. The C. elegans and the human studies together indicate that HBx targets Bcl-2 proteins to promote cytosolic calcium elevation, cell death, and viral replication during HBV infection, which may present an excellent therapeutic intervention point in treating chronic HBV patients. These studies also validate the use of C. elegans as an animal model of HBV.
We are in the process of mapping and cloning the hids genes. We also initiated a large-scale drug screen in C. elegans to isolate compounds that inhibit HBx-induced cell death using a new drug screen strategy that we developed (Kokel et al. Nature Chemical Biology 2006). These studies will provide important insights into what HBx host targets and signaling pathways are and how HBx causes cell death and promotes viral replication and pathogenesis in hepatocytes.
1. Geng, X., Harry, B.L., Zhou, Q.H., Skeen-Gaar, R.B., Ge, X., Lee, E.S., Mitani, S., and Xue, D. (2012). Hepatitis B Virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in C. elegans. Proc. Natl. Acad. Sci. USA 109: 18465-18470. (Abstract and PDF)
2. Geng, X., Huang, C.H., Qin, Y., McComb, J., Yuan, Q., Harry, B.L., Palmer, A., Xia, N.S., and Xue, D. (2012). Hepatitis B virus X protein targets Bcl-2 proteins to increase cytosolic Ca2+, required for virus replication and cell death induction. Proc. Natl. Acad. Sci. USA 109, 18471-18476. (Abstract and PDF)