Nucleotide 880 splice donor site required for efficient transformation and RNA accumulation by human papillomavirus type 16 E7 gene.
Mutations within coding sequences of the various human papillomavirus type 16 (HPV-16) genes have been used to demonstrate that the HPV-16 E7 gene is necessary and sufficient for transformation of rodent cells. We now provide evidence that, in addition to E7 coding sequences, a small cis-acting region immediately flanking the 3' end of E7 coding sequences is also required for transformation. This was shown by translation termination linker insertion, progressive deletion analysis, and site-directed mutagenesis. Disruption of the nucleotide (nt) 880 splice donor site within the 3'-flanking region by deletion of as few as 4 nt or substitution of 3 nt totally abolished transformation. Regeneration of the wild-type sequence in a previously transformation-incompetent splice site mutant restored transformation. Mutating the wild-type splice donor site to the consensus splice site resulted in a stronger transformation phenotype, while mutating the +2 position of the consensus sequence significantly reduced the frequency of transformation. It was shown with RNase protection assays that the amount of E7 mRNA in transformation-deficient splice site mutants was much lower. Nuclear runoff experiments revealed that there was no change in the rate of synthesis of E7 message in the nt 880 splice site mutant. Furthermore, mutations of HPV-16 sequences indicated that the two other early region splice donor sites have no more than minor roles in transformation and efficient RNA accumulation. These results indicate that the specific integrity of the nt 880 splice donor site is essential for both accumulation of E7 RNA and efficient E7-mediated transformation.
Belaguli
N S
NS
Division of Basic Medical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
Pater
M M
MM
Pater
A
A
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002472:Cell Transformation, Viral / Q000235:genetics*; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; C059731:oncogene protein E7, Human papillomavirus type 16