The long-term objectives of this proposal are to understand the role of the hepatitis B virus (HBV)HBx gene in viral infection, and its possible contribution to development of hepatocellular carcinoma (HCC) during chronic HBV infection.
In Aim 1 the molecular mechanism for HBx activation of Ras will be investigated. HBx activation of Ras, and its downstream signalling pathways, represents one of the few HBx activities for which there is reasonable agreement. Activation of Ras by HBx could obviously play an important role in both viral infection and development of HCC. Studies are outlined to explore the molecular basis for HBx activation of Ras, since this could provide important insights into the role of HBx in infection and possibly in carcinoma.
In Aim 2 we will probe the role of HBx in viral infection and carcinogenesis. Until recently, probing the role of HBx in viral infection had been an almost intractable problem. However, models have recently been developed to examine the role of HBx in transformation using transgenic mice and cultured cells. Other model systems have also been developed to permit authentic hepadnaviral replication in transduced rodent liver in vivo, and in vitro in primary human hepatocyte cultures from explanted human liver. HBV and WHV viruses deleted of HBx gene function can therefore be complemented in trans with wildtype and mutant HBx genes to address, for the first time, key questions regarding the role of HBx in viral infection and carcinogenesis in biologically relevant systems. In addition, two HBx activities have been separated by mutagenesis: nuclear transcriptional activation of HBV promoters, and cytoplasmic activation of Ras. Studies will therefore assess which HBx activities, nuclear transcriptional activation, or cytoplasmic activation of Ras signalling pathways (or both), are involved in viral infection and carcinogenesis.
In Aim 3, studies are proposed to study the mechanism and biological significance of HBx mediated cell death. It is generally well established that when expressed at high levels, HBx protein is toxic (lethal) to cells. Results are presented consistent with HBx induction of apoptosis at high levels of expression, and potentiation of apoptosis by TNF (an immune cytokine that induces apoptosis) at low levels of expression, much as described for the HIV Tat protein. Studies will rigorously explore whether HBx induces cell death apoptotically, and if so, the mechanism of induction and the biological implications for viral infection, disease pathogenesis and the process of transformation.
|Puro, Robyn; Schneider, Robert J (2007) Tumor necrosis factor activates a conserved innate antiviral response to hepatitis B virus that destabilizes nucleocapsids and reduces nuclear viral DNA. J Virol 81:7351-62|
|Biermer, Michael; Puro, Robyn; Schneider, Robert J (2003) Tumor necrosis factor alpha inhibition of hepatitis B virus replication involves disruption of capsid Integrity through activation of NF-kappaB. J Virol 77:4033-42|
|Bouchard, Michael J; Puro, Robyn J; Wang, Lihua et al. (2003) Activation and inhibition of cellular calcium and tyrosine kinase signaling pathways identify targets of the HBx protein involved in hepatitis B virus replication. J Virol 77:7713-9|
|Bouchard, M; Giannakopoulos, S; Wang, E H et al. (2001) Hepatitis B virus HBx protein activation of cyclin A-cyclin-dependent kinase 2 complexes and G1 transit via a Src kinase pathway. J Virol 75:4247-57|
|Su, F; Schneider, R J (1996) Hepatitis B virus HBx protein activates transcription factor NF-kappaB by acting on multiple cytoplasmic inhibitors of rel-related proteins. J Virol 70:4558-66|
|Benn, J; Su, F; Doria, M et al. (1996) Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases. J Virol 70:4978-85|
|Benn, J; Schneider, R J (1995) Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls. Proc Natl Acad Sci U S A 92:11215-9|
|Doria, M; Klein, N; Lucito, R et al. (1995) The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J 14:4747-57|
|Benn, J; Schneider, R J (1994) Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci U S A 91:10350-4|