Mutational analysis of human papillomavirus type 16 E7 functions.
The human papillomavirus type 16 E7 gene encodes a nuclear oncoprotein (98 amino acids [AAs] long) consisting of three regions: regions 1 (AAs 1 to 20) and 2 (AAs 21 to 40), which show high homology to the sequences of conserved domains 1 and 2, respectively, of adenovirus E1A; and region 3 (AAs 41 to 98) containing two metal-binding motifs Cys-X-X-Cys (AAs 58 and 91 to 94). We constructed AA deletion (substitution) mutants and single-AA substitution mutants of E7 placed under the control of the simian virus 40 promoter and examined their biological functions. Stable expression of E7 protein in monkey COS-1 cells required almost the entire length of E7 and was markedly lowered by the mutations in region 3. Transactivation of the adenovirus E2 promoter in monkey CV-1 cells was lowered by the mutations. It was abolished by changing Cys-24 to Gly and markedly decreased by a mutation at His-2 or at the metal-binding motifs in region 3. Focal transformation of rat 3Y1 cells by E7 was eliminated by changing His-2 to Asp or Cys-24 to Gly and was greatly impaired by changing Cys-61 or Cys-94 to Gly. The transforming function survived mutations at Leu-13 and Cys-68 and deletion of Asp-Ser-Ser (AAs 30 to 32). The data suggest that regions 1 to 3 are required for its functions and that the meta-binding motifs in region 3 are required to maintain a stable or functional structure of the E7 protein.
Watanabe
S
S
Department of Enteroviruses, National Institute of Health, Tokyo, Japan.
Kanda
T
T
Sato
H
H
Furuno
A
A
Yoshiike
K
K
D000260:Adenoviruses, Human / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002872:Chromosome Deletion; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D027383:Papillomaviridae / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics*
D014764:Viral Proteins; D015678:Viral Structural Proteins