Amino acids of Epstein-Barr virus nuclear antigen 3A essential for repression of Jκ-mediated transcription and their evolutionary conservation

Epstein-Barr virus (EBV) nuclear antigen 3A (EBNA-3A) is essential for virus-mediated immortalization of B lymphocytes in vitro and is believed to regulate transcription of cellular and/or viral genes. One known mechanism of regulation is through its interaction with the cellular transcription factor J kappa, This interaction downregulates transcription mediated by EBNA-2 and J kappa. TO identify the amino acids that play a role in this interaction, we have generated mutant EBNA-3A proteins. A mutant EBNA-3A protein in which alanine residues were substituted for amino acids 199, 200, and 202 no longer downregulated transcription. Surprisingly, this mutant protein remained able to coimmunoprecipitate with J kappa, Using a reporter gene assay based on the recruitment of J kappa by various regions spanning EBNA-3A, we have shown that this mutation abolished binding of J kappa to the N-proximal region (amino acids 125 to 222) and that no other region of EBNA-3A alone was sufficient to mediate an association with J kappa. To determine the biological significance of the interaction of EBNA-3A with J kappa, we have studied its conservation in the simian lymphocryptovirus herpesvirus papio (HVP) by cloning HVP-3A, the homolog of EBNA-3A encoded by this virus. This 903-amino-acid protein exhibited 37% identity with its EBV counterpart, mainly within the amino-terminal half. HVP-3A also interacted with J kappa through a region located between amino acids 127 and 223 and also repressed transcription mediated through EBNA-2 and J kappa, The evolutionary conservation of this function, in proteins that have otherwise significantly diverged, argues strongly for an important biological role in virus-mediated immortalization of B lymphocytes.