Determination of the region of hepatitis B virus X protein required for activation of mitogenic pathways
The Hepatitis B virus X protein is a dual activator of transcription, acting by activating cellular mitogenic pathways and by increasing the transcriptional efficacy of CREB/ATF proteins. The region of pX spanning a.a. 51-140 is sufficient for CREB/ATF trans-activation. However, the region of pX required for mitogenic pathway activation has not been conclusively determined. Since the activation by pX of the cellular mitogenic pathways converges on the activation of the CREB/ATF downstream effectors, I formulated the hypothesis that the region of pX required for mitogenic pathway activation overlaps the region of pX required for CREB/ATF-mediated transcription. To test this hypothesis, I defined the pX region required for mitogenic pathway activation, by cloning a series of X deletions, which were previously studied in our CREB/ATF assays, into a tetracycline-regulated vector and constructing the corresponding clonal cell lines in the less differentiated AML12 clone 4 cell line. Employing these cell lines, I demonstrate that X51-140 is also required for mitogenic pathway activation by monitoring: (a) the pX-dependent activation of JNK, p38 and Ras-Raf-MAPK pathways in transient trans-reporter assays; and (b) the pX-dependent expression of the endogenous cyclin A gene, employing real-time PCR. Interestingly pX deletions lacking a.a. 1-50 or 1-78 display enhanced mitogenic pathway activation, suggesting that this N-terminal pX region acts as a negative modulator of pX function. To test this hypothesis, I have constructed retroviral vectors expressing the N-terminal X1-50 and X1-78 regions. I demonstrate that expression of the N-terminal X region interferes with X-mediated activation of mitogenic pathways and endogenous cyclin A expression. To understand how the N-terminal region of X interferes with WT X function, I employed two tetracycline-regulated cell lines expressing X1-140 and X51-140. Expression of the N-terminal X1-78 interferes only with the function of X 1-140. Moreover, X1-78 co-immunoprecipitates with WT pX, supporting direct protein-protein interaction between the N-terminal regions of two X protein molecules. I conclude that the region of pX spanning a.a 51-140 harbors both the trans-activation function and the mitogenic pathway activation function of pX. In addition, I have identified a region located in the N-terminal portion of pX, spanning a.a. 1-78, which modulates X protein function.
Andrisani, Purdue University.
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