Conserved regions in the Epstein-Barr virus leader protein define distinctdomains required for nuclear localization and transcriptional cooperation with EBNA2

Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood, Recent studies have shown that EBNA-LP may be an importan t EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introd uced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the la tency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are locat ed in the repeat domains that are essential for the EBNA2 cooperativity fun ction, In addition, three serine residues are also well conserved in the re peat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA- LP with two repeats, which is the minimal protein able to cooperate with EB NA2, Mutations introduced into CR1a had no effect on EBNA-LP function, whil e mutations introduced into CR1b resulted in EBNA-LP with slightly decrease d activity, Mutations in CR1c and CR2 resulted in proteins that no longer l ocalized exclusively to the nucleus and also had no EBNA2 cooperation activ ity, Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved s erines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP prote ins with substantially reduced activity, The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleoti des encoding these positively charged amino acid groupings into a cytoplasm ic test protein, herpes simplex virus Delta IE175, and by examining the int racellular localization of the resulting proteins, This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (1 09 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function indepen dently as a nuclear localization signal. However, a combination of amino ac ids 29 to 50 resulted in more efficient nuclear localization than with amin o acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is ess ential for EBNA2 cooperativity function, Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explana tion for why this isoform has no activity. In addition, two conserved serin e residues which are distinct from nuclear import functions are important f or EBNA2 cooperativity function.