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Human papillomavirus (HPV) is frequently associated with cervical carcinoma and derived cell lines. In primary tissues of the carcinoma, the viral genome may be present in episomal or integrated configuration. In cell lines, however, only integrated HPV sequences have been reported. In this article, we describe the presence of episomal type 16 HPV (HPV16), demonstrated by electron microscopy and two-dimensional agarose gel electrophoresis, in a cervical carcinoma cell line, CC7T/VGH, established in 1980 in Taiwan. In CC7T/VGH, the HPV16 sequences are transcriptionally active, and at least three major HPV16 RNA species were detected in Northern blots. Results from restriction enzyme and S1 nuclease analysis suggest a composition of oligomeric HPV16 molecules in dimeric repeats. In addition, the HPV16 oligomers exist as catenated molecules of interlocking rings instead of concatemers. A monomeric copy of the HPV16 episome was cloned from a Hirt supernatant of CC7T/VGH by using a plasmid vector. Mapping and partial sequencing studies revealed an internal deletion of 163 base pairs within the L1 open reading frame. However, insertion of an A.C nucleotide pair at the deletion junction restored the otherwise frame-shifted L1 open reading frame. Two base transitions were also found within the E7 and the E1 open reading frames. Our findings suggest the need for closer examination for HPV episomal catenation in other cervical carcinoma cell lines as well as in primary carcinoma tissues of the uterine cervix and the anogenital tract. With CC7T/VGH, a way is now available for studies of many important aspects of the biology of HPV such as replication and gene expression of the extrachromosomal viral genome.

Integrated human papillomavirus type 16 (HPV16) sequences were cloned from a cervical carcinoma and analyzed by restriction mapping and nucleotide sequencing. The viral integration sites were mapped within the E1 and E2 open reading frames (ORFs). The E4 and E5 ORFs were entirely deleted. An internal deletion of 376 base pairs (bp) was found disrupting the L1 and L2 ORFs. Sequencing analysis showed that an AGATGT/ACATCT inverted repeat marked the deletion junction with two flanking direct repeats 14 and 8 bp in length. A 1,330-bp sequence duplication containing the long control region (LCR) and the E6 and E7 ORFs was also found. The duplication junction was formed by two 24-bp direct repeats with 79% (19 of 24) homology located within the LCR and the E2 ORF of the prototype viral genome, respectively. This observation leads us to propose that the initial viral integration involved an HPV16 dimer in which the direct repeats in tandem units recombined, resulting in reiteration of only a portion of the original duplication. A guanosine insertion between nucleotides 1137 and 1138 created a continuous E1 ORF which was previously shown to be disrupted. Results from this study indicate that sequence reiteration and internal deletion in the integrated, and possibly in the episomal, HPV16 genome are influenced by specific nucleotide sequences in the viral genome. Moreover, reiteration of the LCR/E6/E7 sequences further supports the hypothesis that the E6/E7 ORFs may code for oncogenic proteins and that regulatory signals in the LCR may play a role in cellular transformation.