Mutational analysis of human papillomavirus type 16 E6 protein: transforming function for human cells and degradation of p53 in vitro.
The E6 oncoprotein of human papillomavirus type 16 (HPV 16) [151amino acids (AA) long] contains four metal-binding motifs, C-X-X-C, and is postulated to form two 29-AA finger-like structures in the N-terminal and C-terminal halves, which mediate degradation of p53 and binding to p53, respectively. We constructed a series of E6 mutants with single-AA substitutions in these finger regions (AAs 34-62 and 107-135) and examined their transforming function for human embryonic kidney (HEK) cells in conjunction with HPV 16 E7 and their interaction with human p53 in vitro. The mutants with substitution of L for F-37, G for L-50, S for Y-54, and P for L-110, which did not transform HEK cells, showed markedly lowered activity to direct degradation of p53. The mutants with substitutions of G for R-39, G for V-42, G for Y-43, L for F-47, and G for V-53 lost the transforming function, but they could mediate degradation of p53 at levels comparable to the activities of the wild-type and transforming mutants. Like the wild type, all of the E6 mutants were localized by immunofluorescence to the nuclei of human TS21B cells or monkey COS-1 cells, except for the E6 mutant with substitution of G for Y-43 whose expression was undetectable. The levels of E6 mutants metabolically labeled in COS-1 cells were comparable to those of the transforming E6s. The data indicate that E6-directed degradation of p53 is necessary but not sufficient for HPV 16-mediated transformation of of HEK cells.
Nakagawa
S
S
Division of Molecular Genetics, National Institute of Health, Tokyo, Japan.
Watanabe
S
S
Yoshikawa
H
H
Taketani
Y
Y
Yoshiike
K
K
Kanda
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000821:virology; D002472:Cell Transformation, Viral / Q000502:physiology*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000821:virology; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000821:virology; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009856:Oncogene Proteins, Viral / Q000032:analysis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D027383:Papillomaviridae / Q000235:genetics / Q000502:physiology*; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology*
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; C059731:oncogene protein E7, Human papillomavirus type 16