EBNA-2 OF HERPESVIRUS PAPIO DIVERGES SIGNIFICANTLY FROM THE TYPE-A AND TYPE-B EBNA-2 PROTEINS OF EPSTEIN-BARR-VIRUS BUT RETAINS AN EFFICIENT TRANSACTIVATION DOMAIN WITH A CONSERVED HYDROPHOBIC MOTIF

EBNA-2 contributes to the establishment of Epstein-Barr virus (EBV) la tency in B cells and to the resultant alterations in B-cell growth pat tern by up-regulating expression from specific viral and cellular prom oters. We have taken a comparative approach toward characterizing func tional domains within EBNA-2. To this end, we have cloned and sequence d the EBNA-2 gene from the closely related baboon virus herpesvirus pa pio (HVP). All human EBV isolates have either a type A or type B EBNA- 2 gene. However, the HVP EBNA-2 gene falls into neither the type A cat egory nor the type B category, suggesting that the separation into the se two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identit y with EBV type A EBNA-2 and 35% amino acid identity with type B EBNA- 2. To define the domains of EBNA-2 required for transcriptional activa tion, the DNA binding domain of the GALA protein was fused to overlapp ing segments of EBV EBNA-2. This approach identified a 40-amino-acid ( 40-aa) EBNA-2 activation domain located between aa 437 and 477. Transa ctivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 4 37 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. T he aa 437 boundary identified in these experiments coincides precisely with a block of conserved sequences in HVP EBNA-2, and the comparable carboxy-terminal region of HVP EBNA-2 also functioned as a strong tra nscriptional activation domain when fused to the Gal4(1-147) protein. The EBV and HVP EBNA-2 activation domains share a mixed proline-rich, negatively charged character with a striking conservation of positiona lly equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutage nesis. Any alteration of these residues was found to reduce transactiv ation efficiency, with changes at the Pro-437 and Phe-440 positions pr oducing the most deleterious effects. Activation of the EBV latency C promoter by EBNA-2 was shown to be dependent on the presence of the ca rboxy-terminal activation domain. However, this requirement was generi c, rather than specific, since the EBNA-2 activation domain could be r eplaced with those from the herpes simplex virus (HSV) VP16 protein or the EBV Rta protein. Potential karyophilic signals within EBNA-2 were examined by introducing oligonucleotides encoding positively charged amino acid groupings that might serve in this capacity into a cytoplas mic test protein, HSV DELTAIE175, and by examining the intracellular l ocalization of the resulting proteins. This assay identified a strong nuclear localization signal between EBV amino acids (aa) 478 to 485, w hich was conserved in HVP, and a weaker noncanonical signal between EB V aa 341 to 355, which was not conserved in HVP.