Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the exp ression of viral and cellular genes required for EBV-driven B-cell immortal ization. Elucidating the mechanisms by which EBNA2 regulates viral and cell ular gene expression is necessary to understand EBV-induced B cell immortal ization and viral latency in humans. EBNA2 targets to the latency C promote r (Cp) through an interaction with the cellular DNA binding protein CBF1 (R BPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promo ter binding factor 2 (CBF2), whose protein product(s) has not yet been iden tified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this elemen t is required for efficient activation of Cp by EBNA2. In this study, the C BF2 activity was biochemically purified and microsequenced. The peptides se quenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 b ut not antibodies to related hnRNP proteins reacted with CBF2 in gel mobili ty shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP) /protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. F urthermore, the CBF2 binding site was able to confer EBNA2 responsiveness t o a heterologous promoter when transfected cells were treated with compound s that activate PKA or by cotransfection of plasmids expressing a constitut ively active catalytic subunit of PKA, EBNA2-mediated stimulation of the la tency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivati on; they identify the hnRNP protein AUF1 as a major component of CBF2 and a re also the first evidence of a cis-acting sequence other than a CBF1 bindi ng element that is able to confer responsiveness to EBNA2.