Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa

Abstract

Human papillomavirus (HPV) 16 is frequently found integrated into cervical cancer cell genomes and these integrations are thought to play a role in tumorigenesis. To investigate the mechanisms of HPV integration and its effect on transcription and chromosomal sequence organization, we have cloned and analyzed the HPV16 integration from the cervical cancer cell line SiHa. Restriction analysis and Southern blotting indicated that approximately 95% of an HPV16 genome was integrated without gross rearrangement. Sequence analysis of the cellular-viral DNA junctions revealed that integration had occurred within the E2 and E4 ORFs where 251 bp of viral sequence was deleted. One viral terminus occurred within sequences of an Alu repeat and a 4-bp homology was present at the site of recombination. Using unique cellular flanking DNA probes, a 4.8-kb deletion of cellular sequences was detected at the site of viral integration. The chromosomal location of the viral integration and cellular deletion were mapped to chromosome 13 using a rodent × human somatic cell hybrid panel. Northern blot analysis using viral subgenomic and 3′ cellular probes revealed transcription from the 3′ portion of integrated HPV16 (E6, E7, E1) and flanking cellular sequences. The observation of viral-cell transcripts and chromosomal deletions associated with HPV integration may indicate that such events are part of a multistep mechanism leading to the development of cervical cancer.