Epstein-Barr virus BamHI-A rightward transcript-encoded RPMS protein interacts with the CBF1-associated corepressor CIR to negatively regulate the activity of EBNA2 and NotchIC

The Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) are expr essed in all EBV-associated tumors as well as in latently infected B cells in vivo and cultured B-cell lines. One of the BART family transcripts conta ins an open reading frame, RPMS1, that encodes a nuclear protein termed RPM S. Reverse transcription-PCR analysis revealed that BART transcripts with t he splicing pattern that generates the RPMS1 open reading frame are commonl y expressed in EBV-positive lymphoblastoid cell lines and are also detected in Hodgkin's disease tissues. Experiments undertaken to determine the func tion of RPMS revealed that RPMS interacts with both CBF1 and components of the CBF1-associated corepressor complex. RPMS interaction with CBF1 was dem onstrated in a glutathione S-transferase (GST) affinity assay and by the ab ility of RPMS to alter the intracellular localization of a mutant CBF1. A G al4-RPMS fusion protein mediated transcriptional repression, suggesting an additional interaction between RPMS and corepressor proteins. GST affinity assays revealed interaction between RPMS and the corepressor Sin3A and CIR. The RPMS-CIR interaction nas further substantiated in mammalian two-hybrid , coimmunoprecipitation, and colocalization experiments. RPMS has been show n to interfere with NotchIC and EBNA2 activation of CBF1-containing promote rs in reporter assays. Consistent with this function, immunofluorescence as says performed on cotransfected cells showed that there was colocalization of RPMS with NotchIC and with EBNA2 in intranuclear punctate speckles. The effect of RPMS on NotehIC function was further examined in a muscle cell di fferentiation assay where RPMS was found to partially reverse NotchIC-media ted inhibition of differentiation. The mechanism of RPMS action was examine d in cotransfection and mammalian two-hybrid assays. The results revealed t hat RPMS blocked relief of CBF1-mediated repression and interfered with SKI P-CIR interactions. We conclude that RPMS acts as a negative regulator of E ENA2 and Notch activity through its interactions with the CBF1-associated c orepressor complex.