Functional analysis of EA-D of Epstein-Barr virus.

Different mutations were generated in the diffused-form early antigen (EA-D) of Epstein-Barr virus (EBV) for study of the effects of these mutations in DNA binding, stimulating the activity of EBV-specific DNA polymerase (EBV-DP), and binding to monoclonal antibody R3. Results revealed that the N-terminal 303 amino acids were essential for DNA binding and were sufficient for the enhancement of the activity of EBV-specific DNA polymerase. Deletion study also showed that the region recognized by the R3 monoclonal antibody was located between aa 315 and aa 377. Our results failed to demonstrate the binding between EA-D and EBV-DP, using the proteins synthesized in vitro, suggesting that direct contact between the two proteins is not required for the EBV-DP activity in vitro. We have generated fusion between EA-D and DNA-binding domain of yeast GAL4 protein; however, this fusion protein was not able to transactivate the promoter containing UAS sequence in P3HR1 cells.