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Ras- and Raf-dependent activation of c-jun transcriptional activity by the hepatitis B virus transactivator pX
G Natoli1, M L Avantaggiati, P Chirillo
1Istituto di I Clinica Medica, Policlinico Umberto I, Università degli studi di Roma La Sapienza, Italy.
Abstract:
The mechanisms by which pX, the transactivator of the Hepatitis B Virus (HBV), exerts its effects on transcription of viral and cellular genes have not yet been fully clarified. While previous reports suggested the possibility of a direct interaction of pX, which lacks intrinsic DNA-binding activity, with components of the cellular transcription machinery, more recent investigations support the hypothesis that pX might activate cellular kinases involved in transcriptional regulation and growth control. We analysed the mechanisms of c-Jun transcription factor activation by pX and in particular the role of cellular proteins involved in the transduction of mitogenic signals (namely Ha-Ras and Raf-1). In both HeLa and undifferentiated F9 cells pX was able to increase the activity of exogenous transfected c-Jun but not of c-Jun proteins bearing mutations in the serine residues located in the amino-terminal transcriptional activation domain. We show by use of Ha-Ras and Raf-1 dominant negative mutants that both Ha-Ras and Raf-1 are required for pX-induced activation of c-Jun transcriptional activity. In addition we show that pX is able to cooperate with Raf-1 in c-Jun activation. Our results are consistent with the hypothesis that at least one site of action of pX is peripheral and is located upstream of the Ras genes products.
Insights
Hepatitis B Virus pX protein activates c-Jun transcription via the Ras/Raf signaling pathway. This interaction requires specific serine residues on c-Jun and involves Ha-Ras and Raf-1 proteins.
Area of Science:
- Molecular Biology
- Virology
- Cell Signaling
Background:
- The Hepatitis B Virus (HBV) pX protein is a viral transactivator with incompletely understood mechanisms.
- Previous studies suggested direct interaction with transcription machinery or activation of cellular kinases.
- Recent focus is on pX's role in transcriptional regulation and cellular growth control.
Purpose of the Study:
- To elucidate the mechanism of c-Jun transcription factor activation by HBV pX.
- To investigate the role of mitogenic signaling proteins, specifically Ha-Ras and Raf-1, in pX-mediated c-Jun activation.
Main Methods:
- Analysis of c-Jun transcriptional activity in HeLa and F9 cells following pX expression.
- Utilized site-directed mutagenesis of c-Jun to assess the role of serine residues in activation.
- Employed dominant-negative mutants of Ha-Ras and Raf-1 to probe their involvement in the signaling pathway.
Main Results:
- HBV pX enhanced the activity of wild-type c-Jun but not mutated forms lacking functional serine residues.
- Both Ha-Ras and Raf-1 were found to be essential for pX-induced c-Jun activation.
- pX demonstrated cooperative activity with Raf-1 in activating c-Jun.
Conclusions:
- HBV pX activates c-Jun transcriptional activity through the Ha-Ras/Raf-1 signaling cascade.
- The mechanism involves phosphorylation of serine residues in c-Jun's activation domain.
- pX likely acts upstream of Ras pathway components, suggesting a peripheral site of action.