Sequence and functional analysis of EBNA-LP and EBNA2 proteins from nonhuman primate lymphocryptoviruses

The Epstein-Barr virus (EBV) EBNA-LP and EBNA2 proteins are the first to be synthesized during establishment of latent infection in B lymphocytes. EBN A2 is a key transcriptional regulator of both viral and cellular gene expre ssion and is essential for EBV-induced immortalization of B lymphocytes. EB NA-LP is also important for EBV-induced immortalization of B lymphocytes, b ut far less is known about the functional domains and cellular cofactors th at mediate EBNA-LP function. While recent studies suggest that serine phosp horylation of EBNA-LP and coactivation of EBNA2-medliated transactivation a re important, more detailed mutational and genetic studies are complicated by the repeat regions that comprise the majority of the EBNA-LP sequence. T herefore, we have used a comparative approach by studying the EBNA-LP homol ogues from baboon and rhesus macaque lymphocryptoviruses (LCVs) (baboon LCV and rhesus LCV). The predicted baboon and rhesus LCV EBNA-LP amino acid se quences are 61 and 64% identical to the EBV EBNA-LP W1 and W2 exons and 51% identical to the EBV EBNA-LP Y1 and Y2 exons. Five evolutionarily conserve d regions can be defined, and four of eight potential serine residues are c onserved among all three EBNA-LPs. The major internal repeat sequence also revealed a highly conserved Wp EBNA promoter with strong conservation of up stream activating sequences important for Wp transcriptional regulation. To test whether transcriptional coactivating properties were common to the rh esus LCV EBNA-LP, a rhesus LCV EBNA2 homologue was cloned and expressed. Th e rhesus LCV EBNA2 transcriptionally transactivates EBNA2-responsive promot ers through a CBF1-dependent mechanism. The rhesus LCV EBNA-LP was able to further enhance rhesus LCV or EBV EBNA2 transactivation 5- to 12-fold. Thus , there is strong structural and functional conservation among the simian E BNA-LP homologues. Identification of evolutionarily conserved serine residu es and regions in EBNA-LP homologues provides important clues for identifyi ng the cellular cofactors and molecular mechanisms mediating these conserve d viral functions.