Mutational analysis of the E5 protein of human papillomavirus type 16 to analyze the role of interaction with the 16-kDa subunit of the vacuolar H(+)-ATPase in inhibiting activity of this complex and altering metabolism of the epidermal growth factor receptor.

摘要

The E5 gene of human papillomavirus type 16 encodes an 83-amino acid hydrophobic membrane protein that has been demonstrated to upregulate expression of the epidermal growth factor receptor (EGFR) by inhibiting degradation of internalized EGFRs and inducing receptor recycling to the cell surface. E5 also inhibits endosomal acidification, presumably through its interaction with the 16-kDa pore-forming subunit of the vacuolar H+-ATPase (V-ATPase). To explore the relationship between these activities of E5, extensive mutagenesis of E5 was performed to determine the binding site for the 16-kDa protein. These studies revealed that two regions within the C-terminal half of E5 participate in this interaction, with the sequence encompassing residues 41 to 54 being particularly critical. This latter region can be replaced with random hydrophobic amino acids without abrogation of binding, suggesting that the hydrophobicity of this segment and not its primary sequence is critical for binding. Coimmunoprecipitation studies with mutated 16-kDa subunits indicated that E5 binds to the fourth transmembrane domain, whose sequence is identical from species as diverse as yeast and man. This result was significant because recent research from this laboratory has demonstrated that expression of E5 in the yeast Saccharomyces cerevisiae produces phenotypes, such as growth inhibition, that are consistent with disruption of V-ATPase activity. When the E5 mutants were expressed in the same system, some mutants maintaining the ability to interact with the 16-kDa subunit no longer delayed growth. Moreover, one deletion mutant that does not bind to the pore subunit still retained the capacity to inhibit growth. Together, these data suggest that E5 interaction with the 16-kDa protein may be neither necessary nor sufficient to disturb V-ATPase activity. Efforts to detect delayed EGFR degradation in the presence of wild-type E5 were unsuccessful and only a modest increase in activity of the downstream effector mitogen-activated protein kinase was found. These results reveal the possibility that additional biologically relevant targets for E5 remain to be discovered and may be important for understanding the role of this protein in the viral life cycle and pathogenesis.