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) latency in B cells and to the resultant alterations in B-cell growth pattern by up-regulating expression from specific viral and cellular promoters. We have taken a comparative approach toward characterizing functional domains within EBNA-2. To this end, we have cloned and sequenced the EBNA-2 gene from the closely related baboon virus herpesvirus papio (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 category nor the type B category, suggesting that the separation into these two subtypes may have been a recent evolutionary event. Comparison of the predicted amino acid sequences indicates 37% amino acid identity 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 activation, the DNA binding domain of the GAL4 protein was fused to overlapping segments of EBV EBNA-2. This approach identified a 40-amino-acid (40-aa) EBNA-2 activation domain located between aa 437 and 477. Transactivation ability was completely lost when the amino-terminal boundary of this domain was moved to aa 441, indicating that the motif at aa 437 to 440, Pro-Ile-Leu-Phe, contains residues critical for function. The 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 transcriptional 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 positionally equivalent hydrophobic residues. The importance of the individual amino acids making up the Pro-Ile-Leu-Phe motif was examined by mutagenesis. Any alteration of these residues was found to reduce transactivation efficiency, with changes at the Pro-437 and Phe-440 positions producing the most deleterious effects. Activation of the EBV latency C promoter by EBNA-2 was shown to be dependent on the presence of the carboxy-terminal activation domain. However, this requirement was generic, rather than specific, since the EBNA-2 activation domain could be replaced 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 cytoplasmic test protein, HSV delta IE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBV amino acids (aa) 478 to 485, which was conserved in HVP, and a weaker noncanonical signal between EBV aa 341 to 355, which was not conserved in HVP.
Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
P D
PD
Ling
J J
JJ
Ryon
S D
SD
Hayward
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D019309:Epstein-Barr Virus Nuclear Antigens; D005455:Fluorescent Antibody Technique; D006564:Herpesviridae / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010215:Papio / Q000382:microbiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics*; D014709:Vero Cells
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D021382:Protein Sorting Signals; D015534:Trans-Activators