Analysis of human papillomavirus type 16 open reading frame E7 immortalizing function in rat embryo fibroblast cells.
The E7 open reading frame of human papillomavirus type 16 (HPV-16) encodes a protein that can immortalize primary rat cells, cooperate with the ras oncoprotein to transform low passage rat cells and transform established rodent cells to anchorage independence. The immortalizing and cooperation functions have been investigated using a series of point mutations that introduce single amino acid changes into the E7 protein in two distinct regions. Certain mutations altering amino acids conserved between the E7 protein of genital HPV types, the adenovirus E1a protein and simian virus 40 large T antigen abolished the ability of the E7 protein to immortalize or cooperate with ras in a focus forming assay. Mutations in a consensus sequence for a casein kinase II recognition site, which is also shared by E1a and large T, reduced immortalizing activity, but did not affect the ability to cooperate with ras. Single mutations disrupting cysteine motifs, which form putative zinc-binding sites in the second region, reduced the activity of the E7 protein, whereas double mutants, in which neither of the cysteine motifs remained intact, showed no or very low activity. The activity of the mutants in immortalization and cooperation assays was essentially the same as their transforming activities in NIH 3T3 cells. This indicates that these three functions of E7 map to overlapping domains which cannot be separated by these mutations in the region of E1a/large T homology or the cysteine motifs.
Chesters
P M
PM
Department of Microbiology, United Medical School, Guy's Hospital, London, U.K.
Vousden
K H
KH
Edmonds
C
C
McCance
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D005347:Fibroblasts; D008969:Molecular Sequence Data; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D051381:Rats; D014162:Transfection
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; C059731:oncogene protein E7, Human papillomavirus type 16