Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence duplication and internal deletion in the integrated human papillomavirus type 16 genome cloned from a cervical carcinoma.

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.     

Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells.

We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1. Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors. The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter. Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16. By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA. It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA. These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells. Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.     

Human papillomavirus type 6 long control region and human cellular DNA contain related sequences.

We have identified a region of human papillomavirus type 6 (HPV-6) DNA that hybridizes with human cellular DNA containing no detectable HPV DNA sequences. The region of hybridization has been localized to a segment of the viral long control region between the end of the L1 open reading frame and the late polyadenylation signal and is likely contained within a 94-base-pair insertion at nucleotide 7350 which is present in the cloned HPV-6b DNA used for these studies. Restriction fragments of HPV-6 DNA from seven patients suggested that this insert was present in these naturally occurring viral genomes as well. The presence of this insert was confirmed by direct sequence analysis of polymerase chain reaction-amplified segments from four naturally occurring HPV-6 genomes. By analogy with other systems, this insert and surrounding sequences may function to destabilize the HPV-6 late mRNA.     

High sensitivity detection of HPV-16 in SiHa and CaSki cells utilizing FISH enhanced by TSA

 Detection of integrated human papillomavirus type 16 (HPV-16) DNA in SiHa and CaSki cells was used as a model system to demonstrate sensitivity and resolution of a well defined target. Using 293- to 1987-base polymerase chain reaction (PCR)-synthesized probes to the E6 and E7 open reading frames of HPV-16, several fluorescent in situ hybridization (FISH) detection methods, enhanced with tyramide signal amplification (TSA), were compared. The synthetic probes were biotin labeled by a nick translation method and the hybridized probes were detected by various fluorescent TSA methods using cyanine 3 tyramide, biotinyl tyramide and a biotin TSA Plus reagent. High sensitivity detection in SiHa cells was demonstrated using a 619-base probe to detect two single copies of integrated HPV-16 DNA. In CaSki cells, which contain up to 600 copies of HPV-16 DNA, a 293-base probe was used for detection. The results of these comparisons show that with refinement of TSA methods and reagents, increasing levels of high sensitivity detection can be achieved and that these methods allow subnuclear localization as well. Accepted: 20 June 1997