Identification and Functional Characterization of Phosphorylation Sites of the Human Papillomavirus 31 Protein

The papillomavirus E2 protein regulates transcription, replication, and nuclear retention of viral genomes. Phosphorylation of E2 in the hinge region has been suggested to modulate protein stability, DNA- binding activity, and chromosomal attachment. The papillomavirus E8<^>E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. Mass spectrometry analyses of human papillomavirus 31 (HPV31) E8<^>E2 and E2 proteins identify phosphorylated S78, S81, and S100 in E8<^>E2 and S266 and S269 in E2 in their hinge regions. Phos- tag analyses of wild- type and mutant proteins indicate that S78 is a major phosphorylation site in E8<^>E2, but the corresponding S266 in E2 is not. Phosphorylation at S78 regulates E8<^>E2's repression activity of reporter constructs, whereas the corresponding E2 mutants do not display a phenotype. Phosphorylation at S78 does not alter E8<^>E2's protein stability, nuclear localization, or binding to DNA or to cellular NCoR/ SMRT complexes. Surprisingly, in the context of HPV31 genomes, mutation of E8<^>E2 S78 does not modulate viral replication or transcription in undifferentiated or differentiated cells. However, comparative transcriptome analyses of differentiated HPV31 E8<^>E2 S78A and S78E cell lines reveal that the expression of a small number of cellular genes is changed. Validation experiments suggest that the transcription of the cellular LYPD2 gene is altered in a phospho- S78 E8<^>E2-dependent manner. In summary, our data suggest that phosphorylation of S78 in E8<^>E2 regulates its repression activity by a novel mechanism, and this seems to be important for the modulation of host cell gene expression but not viral replication. IMPORTANCE Posttranslational modification of viral proteins is a common feature to modulate their activities. Phosphorylation of serine residues S298 and S301 in the hinge region of the bovine papillomavirus type 1 E2 protein has been shown to restrict viral replication. The papillomavirus E8<^>E2 protein shares the hinge domain with E2 and acts as a repressor of viral replication. A large fraction of HPV31 E8<^>E2 is phosphorylated at S78 in the hinge region, and this is important for E8<^>E2's repression activity. Surprisingly, phosphorylation at S78 in E8<^>E2 has no impact on viral replication in tissue culture but rather seems to modulate the expression of a small number of cellular genes. This may indicate that phosphorylation of viral transcription factors serves to broaden their target gene specificity.