A mutagenic analysis of the E5 protein of human papillomavirus type 16 reveals that E5 binding to the vacuolar H+-ATPase is not sufficient for biological activity, using mammalian and yeast expression systems.
The E5 gene of human papillomavirus type 16 encodes a highly hydrophobic membrane protein previously shown to inhibit endosomal acidification, presumably by binding to the 16-kDa pore-forming subunit of the vacuolar H(+)-ATPase (v-ATPase). The role of this interaction in the disruption of v-ATPase activity was explored through extensive mutagenesis of E5 to identify residues that mediate binding to the 16-kDa subunit. Coimmunoprecipitations revealed that the hydrophobic span between residues 41 and 54 is primarily responsible for this interaction and can be replaced with random hydrophobic amino acids. Studies using mutated 16-kDa proteins indicated that the fourth transmembrane domain of the pore subunit mediates binding to E5. Analysis of the E5 mutants in a yeast expression system revealed that several mutants that retained the capacity to bind to the 16-kDa subunit in COS-1 cells failed to disrupt vacuolar acidification. These data argue that E5 binding to the pore subunit is not sufficient for the associated activity of disruption of v-ATPase function.
Adam
J L
JL
Department of Microbiology and Immunology, University of Rochester, Rochester, New York, 14642, USA.
Briggs
M W
MW
McCance
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011510:Proteolipids / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D025262:Vacuolar Proton-Translocating ATPases; D000086082:Viroporin Proteins
D009856:Oncogene Proteins, Viral; D011505:Protein-Tyrosine Kinases; D011510:Proteolipids; D017494:Proton Pumps; D006180:Proton-Translocating ATPases; D025262:Vacuolar Proton-Translocating ATPases; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins