The integration of HPV-18 into HeLa cells has involved duplication of part of the viral genome as well as human DNA flanking sequences.

We have cloned two restriction enzyme fragments from the Hela cell line S3 which hybridize with the genomes of HPV-6 and 16 under conditions of reduced stringency (Tm-43°C). One of the fragments, a 5.7-kb Bam HI fragment, was found to be present within the other, a 15.8-kb Eco RI fragment. Restriction enzyme (RE) mapping of this 15.8-kb fragment (see Figure) revealed that the sequence of RE sites at its right hand end is identical to previously proposed maps of the integration site of HPV-18 in the Hela cell genome". Thus, the 7.0-kb Eco Rl/Hind III and 5.7-kb Hind HI fragments shown in the Figure below, appear to correspond directly to the 8.4 or 7.9-kb and 5.8-kb Hind III fragments identified by Lazo. The RE map also revealed the presence of a 5 to 6-kb region of duplication within the 15.8-kb fragment adjacent to the HPV-18 sequences. Thus, the sequence of RE sites (Pstl, Kpnl, Smal, Hind III) within the 5.7-kb Bam HI fragment is repeated exactly in the 6.6-kb Bam Ul/Eco RI fragment. Individual sub-fragments from the 15.8-kb Eco RI fragment were therefore isolated and used as probes to investigate the nature of this duplication event. Sub-fragments were also probed with HPV-6 and 16 at low stringency and with human genomic DNA at high stringency (Tm-12°C) to determine the distribution of HPV and human genomic sequences within the 15.8-kb fragment. The results of these experiments are shown diagrammatically in the accompanying figure. The approximate extent of the direct repeat unit is also indicated. When the 2.1-kb Hind III fragment and 2.2-kb Bam Hi/Hind III fragment were used to probe Eco RI digests of human genomic DNA, hybridization was visible smearing over the entire range of fragment sizes from 0.5to 23-kb. We interpret this to mean that these fragments contain human repetitive DNA sequences. The 1.35-kb Pstl/Smal and 1.0-kb Sma I/Hind III fragments from both copies of the repeat and the 0.9-kb Hind III fragment from the left end of the 15.8-kb fragment, all hybridized to a single 8.1-kb Eco RI fragment from the human SiHa cell line under conditions of high stringency (Tm-12°C). Also, the 1.1-kb Bam HI fragment (Upstream Regulatory Region/non-coding region) hybridized to the 2.2-kb Bam Hi/Hind III fragment which contains human repetitive DNA sequences. These findings indicate that the integration of HPV-18 into the Hela cell genome not only involved the deletion of viral sequences but also the duplication of viral as well as human repetitive and non-repetitive DNA flanking sequences. The integration of HPV-18 DNA into Hela cells has also been mapped by in situ hybridization.