Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
J Virol. 2020 Jul 1;94(14). doi: 10.1128/JVI.00488-20.
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. 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.
乳头瘤病毒 (PV) E2 蛋白是病毒转录和基因组复制的关键调节剂。我们之前报道过，HPV-31 E2 的酪氨酸 (Y) 138 被成纤维细胞生长因子受体 3 (FGFR3) 激酶磷酸化。在这项研究中，我们生成了包含 G418 可选择的 HPV-31 基因组的准病毒，这些基因组含有磷酸化缺陷的苯丙氨酸突变 E2 Y138F 和磷酸模拟的谷氨酸突变 Y138E。我们观察到，即使 E2 在病毒起源处的占据与野生型 E2 相当，用这些 Y138 突变基因组感染后，早期病毒转录本的数量明显减少。感染 Y138F 准病毒的角质形成细胞形成稳定的集落，基因组作为附加体维持，而感染 Y138E 准病毒的细胞则不能。我们之前报道过，HPV-31 E2 Y138 突变为谷氨酸后不再与 Brd4 C 端基序 (CTM) 结合。在这里，我们证明 HPV-16 E2 Y138E 与全长 Brd4 结合，但不与 Brd4 CTM 结合。我们得出结论，E2 与 Brd4 CTM 的结合对于病毒基因组复制是必要的，并表明这种相互作用可以通过 E2 Y138 的磷酸化来调节。乳头瘤病毒 (PV) 是一种感染皮肤和黏膜上皮的双链 DNA 肿瘤病毒。PV E2 蛋白与许多细胞因子结合，介导 HPV 基因组的复制。成纤维细胞生长因子受体 3 (FGFR3) 通过 HPV E2 蛋白中酪氨酸 138 的磷酸化来调节 HPV 的复制。通过使用准病毒感染模型和 G418 抗性基因组的选择，我们证明 Y138 是 Brd4 结合的关键残基，并且不能与 Brd4 复合不能支持附加体复制。
