Biologic activity of human papillomavirus type 16 E6/E7 cDNA clones isolated from SiHa cervical carcinoma cell line.

Abstract

Three species of E6/E7 cDNAs of human papillomavirus type 16 (HPV16) for the full-length E6/E7 and spliced E6*I/E7 and E6*II/E7 mRNAs were synthesized by reverse transcriptase-(RT-)PCR from RNA of the cervical carcinoma cell line SiHa. Two cDNA mutants carrying point mutations in either a splice donor site or acceptor site within the E6 open reading frame were also constructed. These HPV16 E6/E7 cDNAs were cloned under the SV40 enhancer/promoter and the MMTV LTR to examine the activities of ras-collaborative transformation and induction of cellular DNA synthesis, both of which depend on the E7 gene product. The E6*II/E7 cDNA and two mutated cDNAs deficient in the spliced mRNA transcription showed lower levels of both activities than the full-length E6/E7 and the E6*I/E7 cDNA. The rat cell lines carrying each of the E6/E7 cDNAs contained the E6/E7 mRNA species expected. A small amount of E6*I/E7-sized mRNA was transcribed from a splice-donor site mutant of the E6/E7 cDNA, which turned out to be a transcript derived from a cryptic splice donor site six bases upstream from the conventional site. Among NIH3T3 cells carrying one of the above-mentioned E6/E7 cDNAs, the cells expressing E6*I/E7 mRNA [cells carrying cF(wt) and c*I] produced an amount of E7 protein comparable with those carrying the E7 or E6E7 region. These results suggest that the E6*I/E7 is the mRNA that is important for the efficient expression of E7 product from the HPV16 E6/E7 region.