The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for EBV-dependent immortalization of human primary B lymphocytes. Genetic analysis indicated that amino acids 365 to 992 are important for EBV-mediated immortalization of B lymphocytes. We demonstrate that this region of EBNA3C critical for immortalization interacts with prothymosin alpha (ProT␣), a cellular protein previously identified to be important for cell division and proliferation. This interaction maps to a region downstream of amino acid 365 known to be involved in transcription regulation and critical for EBV-mediated transformation of primary B lymphocytes. Additionally, we show that EBNA3C also interacts with p300, a cellular acetyltransferase. This interaction suggests a possible role in regulation of histone acetylation and chromatin remodeling. An increase in histone acetylation was observed in EBV-transformed lymphoblastoid cell lines, which is consistent with increased cellular gene expression. These cells express the entire repertoire of latent nuclear antigens, including EBNA3C. Expression of EBNA3C in cells with increased acetyltransferase activity mediated by the EBV transactivator EBNA2 results in down-modulation of this activity in a dose-responsive manner. The interactions of EBNA3C with ProT␣ and p300 provide new evidence implicating this essential EBV protein EBNA3C in modulating the acetylation of cellular factors, including histones. Hence, EBNA3C plays a critical role in balancing cellular transcriptional events by linking the biological property of mediating inhibition of EBNA2 transcription activation and the observed histone acetyltransferase activity, thereby orchestrating immortalization of EBV-infected cells.Epstein-Barr Virus (EBV) is a human gammaherpesvirus predominantly infecting epithelial cells of the oropharynx and human primary B lymphocytes (41,63). EBV is the etiological agent of infectious mononucleosis and is also associated with various human malignancies, including Burkitt's lymphoma, nasopharyngeal carcinoma, non-Hodgkin's disease, AIDS immunoblastic lymphomas, and lymphoproliferative disease (3,63). Infection of the oropharyngeal epithelium is predominantly a lytic type of infection with the production of progeny virus (33,61,63,73). Infection of human primary B lymphocytes by EBV transforms them into continuously proliferating lymphoblastoid cell lines (LCLs) in vitro (11,29). Recent studies have demonstrated that EBV utilizes two major cellular signaling pathways for transforming B cells, the NOTCH1 signaling pathway and the TNF signaling pathway (6, 34, 57).After initial infection of B lymphocytes, EBV typically establishes a latent infection with the expression of 11 viral transcripts (41, 63). These genes are the six EBV nuclear antigens (EBNAs), three latent membrane proteins (LMPs), and the EBV early RNAs (41). Only a selected number of these genes are necessary for EBV-mediated immortalization of B lymphocytes (65). EBNA2, EBNA3A, EBNA3C, and LMP1 are essential for EBV-induced immorta...