Functional contribution of Epstein-Barr virus BARF1 residues 35-39 to viable cell maintenance under serum deprivation and tumor formation.

Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) is a viral oncogene implicated in epithelial malignancies, but the determinants of its transforming activity remain incompletely defined. We generated BARF1 truncation constructs and targeted mutants to examine the contribution of residues 35-38 and adjacent Ser39 in BALB/c 3T3 fibroblasts using serum deprivation, tumorigenicity, BCL-2 expression, and molecular docking assays. Deletion of residues 35-38 in full-length BARF1 reduced viable cell maintenance under severe serum deprivation, whereas Ser39-to-Ala substitution in the BARF1 1-56 background markedly reduced the viable cell advantage of this fragment. Neither mutant produced detectable tumors during the 28-day observation period. Full-length BARF1 induced tumors in all animals, whereas BARF1 1-56 retained partial tumor-forming activity. BARF1 1-46 maintained higher viable cell numbers under serum-limiting conditions but produced no detectable tumors, suggesting a contribution of residues 47-56 to tumor formation. BCL-2 remained detectable in both non-tumor-forming mutants. Exploratory docking predicted a more favorable score and distinct positioning for the wild-type 35-39 peptide relative to the catalytic subunit of cAMP-dependent protein kinase A (PKA) than for the mutant peptides; however, this analysis does not demonstrate Ser39 phosphorylation or PKA-dependent BARF1 activity. Overall, these findings support a functional contribution of the BARF1 region encompassing residues 35-39 to viable cell maintenance under serum deprivation and tumor formation. Because construct-level BARF1 protein abundance, stability, secretion, and localization were not assessed, the observed phenotypes cannot be attributed solely to the altered residues.