The Human Papillomavirus Type 11 E1∧E4 Protein Is Phosphorylated in Genital Epithelium

Abstract

The most abundant viral transcript in human papillomavirus (HPV) 11-infected xenograft tissue has been shown to encode the E1∧E4 protein. The function of E1∧E4 protein has not been determined. Several potential phosphorylation sequence motifs were identified in the HPV 11 E1∧E4 protein, including potential sites of phosphorylation by mitogen-activated protein kinase (MAPK), cAMP-dependent protein kinase (PKA), casein kinase II, and protein kinase C. To test phosphorylation of the HPV 11 E1∧E4 protein, a soluble maltose binding protein (MBP) fusion was produced in Escherichia coli. Only MAPK and PKA phosphorylated the E1∧E4 protein. Phosphoamino acid analysis showed that one or more threonine residues were phosphorylated by MAPK, and both serine and threonine residues were phosphorylated by PKA. MBP–E1∧E4 mutant proteins were designed to delineate the E1∧E4 phosphoacceptor residues. MAPK was shown to phosphorylate E1∧E4 on threonine 53 within a MAPK consensus phorphorylation sequence motif. PKA was shown to phosphorylate E1∧E4 at two residues: threonine 36 within a consensus motif and serine 44 within a variant of the PKA consensus phosphorylation sequence motif. HPV 11-infected human genital tissue grown as a xenograft in an athymic mouse was labeled with [32P]orthophosphate. Phosphoamino acid analysis of E1∧E4 protein immunoprecipitated from 32P-labeled tissue revealed that both serine and threonine residues were phosphorylated. Analysis by liquid chromatography–mass spectrophotometry was consistent with phosphorylation of residues within the PKA and MAPK phosphorylation sequence motifs. Expression of E1∧E4 protein containing phosphorylation substitution mutations showed that the PKA mutant did not differ from wild-type E1∧E4 protein in intracellular distribution. In contrast, the MAPK mutant did not localize exclusively to the cytoplasm nor did it colocalize with wild-type E1∧E4 protein. We conclude that HPV 11 E1∧E4 protein is phosphorylated in vitro and in vivo. Our data are consistent with phosphorylation of HPV 11 E1∧E4 protein by MAPK and PKA in infected tissue.