Phosphorylation of the Human Papillomavirus E2 Protein at Tyrosine 138 Regulates Episomal Replication

The papillomavirus (PV) E2 protein is a critical regulator of viral transcription and genome replication. We previously reported that tyrosine (Y) 138 of HPV-31 E2 is phosphorylated by the fibroblast growth factor receptor 3 (FGFR3) kinase. In this study, we generated quasiviruses containing G418-selectable HPV-31 genomes with phosphodeficient phenylalanine mutant E2 Y138F and phosphomimetic glutamic acid mutant Y138E. We observed significantly fewer early viral transcripts immediately after infection with these Y138 mutant genomes even though E2 occupancy at the viral origin was equivalent to that of wild-type E2. Keratinocytes infected with Y138F quasiviruses formed stable colonies, and the genomes were maintained as episomes, while those infected with Y138E quasiviruses did not. We previously reported that the HPV-31 E2 Y138 mutation to glutamic acid did not bind to the Brd4 C-terminal motif (CTM). Here, we demonstrate that HPV-16 E2 Y138E bound to full-length Brd4 but not to the Brd4 CTM. We conclude that association of E2 with the Brd4 CTM is necessary for viral genome replication and suggest that this interaction can be regulated by phosphorylation of E2 Y138. IMPORTANCE Papillomavirus (PV) is a double-stranded DNA tumor virus infecting the cutaneous and mucosal epithelium. The PV E2 protein associates with a number of cellular factors to mediate replication of the HPV genome. Fibroblast growth factor receptor 3 (FGFR3) regulates HPV replication through phosphorylation of tyrosine 138 in the HPV E2 protein. Employing a quasivirus infection model and selection for G418 resistant genomes, we demonstrated that Y138 is a critical residue for Brd4 association and that inability to complex with Brd4 does not support episomal replication.