GENOMIC CLONING AND CHARACTERIZATION OF THE NONOCCUPIED ALLELE CORRESPONDING TO THE INTEGRATION SITE OF HUMAN PAPILLOMAVIRUS TYPE-16 DNA INTHE CERVICAL-CANCER CELL-LINE SIHA

Human papillomavirus (HPV) type 16 DNA sequences have been found integ rated into the host cell genome in a large number of cervical tumors a nd cell lines derived therefrom. In this study, we have cloned and ana lyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses rev ealed an approximately 7.8-kb deletion of cellular DNA upon viral inte gration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral i ntegration site showed no significant homology to any known human sequ ences. DNase I mapping experiments on native chromatin demonstrated th e existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration oc curred close to putative regulatory sequences and that recombination w ith host cellular DNA was not followed by a reorganization of the chro matin structure upstream of the integration site, Nuclear run-on and R T-PCR experiments showed HPV-specific transcription spanning the E2, E 4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HP V-16 regulatory region (URR). Taken together, our data suggest that th e cellular DNA region upstream of the HPV-16 integration site in the S iHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR. (C) 1996 Academic Pres s, Inc.