The association between hepatocellular carcinoma (HCC) and chronic infection with the human hepatitis B virus (HBV) has generated considerable interest in understanding how infection with HBV increases the risk for liver cancer. Based on studies in woodchucks infected with the woodchuck hepatitis virus, it is assumed that the X protein (HBx) of all mammalian hepadnaviruses, including human HBV, is essential for viral replication. There is also evidence that HBx contributes to the development of HCC. HBx activates many cellular signaling cascades;however, how this is accomplished is unclear. Most studies that sought to identify the essential role of HBx during HBV replication, and understand how HBx regulates cellular signaling pathways, were performed in liver cell lines derived from liver tumors. The use of immortalized or transformed hepatocytes suffers from its lack of biological relevance compared to authentic hepatocytes. We have demonstrated that a key activity of HBx is regulation of cellular calcium signaling pathways. This proposal aims to understand key activities of HBx using cultures of primary rat hepatocytes and to determine how these activities influence hepatocyte physiology and HBV replication. Studies in AIM 1 will characterize the molecular mechanism of HBx activation of calcium signaling pathways in hepatocytes and how this influences HBV replication. Studies in AIM 2 will focus on the interaction between HBx and mitochondria and how this impacts hepatocyte physiology and HBV replication.
AIM 3 specifically focuses on regulation of apoptosis by HBx expressed alone or during HBV replication. Although the fundamental activities of HBx are likely preserved in hepatocytes as compared to transformed cells, the magnitude, duration and complexity of these activities may differ in primary hepatocytes. Result from studies in primary rat hepatocytes should more accurately reflect the state of an authentic infected cell, clarify the disparate HBx activites that have been reported, and provide a more sophisticated understanding of the role of HBx during HBV replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI064844-04
Application #
7534001
Study Section
Virology - A Study Section (VIRA)
Program Officer
Berard, Diana S
Project Start
2005-12-01
Project End
2010-11-30
Budget Start
2008-12-01
Budget End
2009-11-30
Support Year
4
Fiscal Year
2009
Total Cost
$314,825
Indirect Cost
Name
Drexel University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
002604817
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Rawat, Siddhartha; Bouchard, Michael J (2015) The hepatitis B virus (HBV) HBx protein activates AKT to simultaneously regulate HBV replication and hepatocyte survival. J Virol 89:999-1012
Yang, Bei; Bouchard, Michael J (2012) The hepatitis B virus X protein elevates cytosolic calcium signals by modulating mitochondrial calcium uptake. J Virol 86:313-27
Gearhart, Tricia L; Bouchard, Michael J (2011) The hepatitis B virus HBx protein modulates cell cycle regulatory proteins in cultured primary human hepatocytes. Virus Res 155:363-7
Lizzano, Rebecca A; Yang, Bei; Clippinger, Amy J et al. (2011) The C-terminal region of the hepatitis B virus X protein is essential for its stability and function. Virus Res 155:231-9
Bouchard, Michael J; Navas-Martin, Sonia (2011) Hepatitis B and C virus hepatocarcinogenesis: lessons learned and future challenges. Cancer Lett 305:123-43
Gearhart, Tricia L; Bouchard, Michael J (2010) Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes. Virology 407:14-25
Gearhart, Tricia L; Bouchard, Michael J (2010) The hepatitis B virus X protein modulates hepatocyte proliferation pathways to stimulate viral replication. J Virol 84:2675-86
Clippinger, Amy J; Gearhart, Tricia L; Bouchard, Michael J (2009) Hepatitis B virus X protein modulates apoptosis in primary rat hepatocytes by regulating both NF-kappaB and the mitochondrial permeability transition pore. J Virol 83:4718-31
Clippinger, Amy J; Bouchard, Michael J (2008) Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential. J Virol 82:6798-811
McClain, Stephanie L; Clippinger, Amy J; Lizzano, Rebecca et al. (2007) Hepatitis B virus replication is associated with an HBx-dependent mitochondrion-regulated increase in cytosolic calcium levels. J Virol 81:12061-5