Investigations of sequence variation in human papillomavirus type 16 cervical cancer isolates

Abstract

Human papillomavirus type 16 (HPV 16) is the major causative agent of cervical cancer worldwide, but the clinical aggression displayed by individual HPV 16 positive cancers is highly variable. This might reflect altered expression of the viral oncogenes resulting fiom sequence variations in the important regulatory region of the viral genome, the upstream regulatory region (URR). Initially, this was assessed by amplifying by polymerase chain reaction (PCR) and sequencing the entire URR of 34 cervical cancer isolates from Australia and New Caledonia. Only one isolate was identical to the HPV 16 prototype; one large—scale change (an 81bp duplication), 44 variant nucleotides, and one single base insertion were identified. Most of the variants were of European lineage (82%), whilst smaller proportions of Asian and Asian American lineage isolates were also identified (12% and 6% respectively).

The level of expression of the viral oncogenes, E6 and E7 is controlled by cellular transcription factors (TFs) binding to their response sites in the URR. A major objective of this study was to analyse the effects of URR sequence changes on oncogene promoter activity. Six of the variant URRs, selected mainly on the basis of potentially important sequence changes, were cloned into a luciferase vector. The constructs were transiently transfected into HeLa and HT3 cells and luciferase activities of variants, a surrogate for E6 and E7 expression, were compared with that of the prototype. Promoter activities controlled by three of the five URRs that contained only minor sequence variations were upregulated between three- and eleven-fold, while that mediated by the URR with the large-scale duplication was comparable to the prototype. Attempts were then made to identify specific sequence changes responsible for upregulated promoter activity by PCR—based site-directed mutagenesis. Changes in a yin and yang 1 (YYl) binding site and an overlapping octamer binding factor—1 (Oct-1)/papilloma enhancer binding factor-1 (PEP-1) site both mediated upregulation of the promoter in the isolate with the highest luciferase activity. Neither of these changes affected the binding of their corresponding TFs in electrophoretic mobility shift assays though. A nucleotide substitution in the papillomavirus silencing motif (PSM) partly explained promoter upregulation in the isolate with the second highest luciferase activity. Further analysis revealed that sequence variation in the PSM affected the binding of CCAAT displacement protein (CDP). Sequence variation in other YYl and transcriptional enhancer factor-l (TEF- 1) binding sites did not affect promoter activity in two of the upregulated isolates.

Deregulation of E6/E7 expression is central to HPV-associated malignant conversion. In many cases this is achieved by integration of viral DNA into the host genome, often disrupting the E1 and/or E2 regions. However, HPV 16-associated cancers may carry only episomal HPV. Sequence variation in the URR is believed to account for at least some instances of malignant conversion in the absence of integration. For this reason the physical state of HPV DNA in the 34 isolates was investigated by Southern hybridisation and PCR of the E2 gene. Overall, for the 25 isolates where results could be obtained, 44% were integrated into host chromosomes, 40% were episomal, and 16% contained both integrated and episomal DNA. The physical state of nine of the isolates could not be determined because of specimen inadequacy. The two isolates with the highest promoter activities and the isolate with the large—scale duplication contained only episomal HPV DNA. Twenty of 28 isolates (71%) contained the entire E2 gene. For the eight isolates lacking an entire E2 gene, deletion of the 3’ region (four isolates), the 5’ region (one isolate), the entire E2 gene (two isolates), or the presence of a discontinuous E2 gene (one isolate) was determined by PCRs spanning different lengths of the E2 region.

In addition to sequence variation in the URR, it has also been suggested that some amino acid substitutions in viral coding regions, such as in E2 and E6 could have biological significance. The potential implications of amino acid changes include changes to B or T cell epitopes and alterations to protein function. The E2 gene and the overlapping E4 gene from 13 of the isolates, and the E6 gene from 27 isolates were sequenced. Twenty one from 30, five from 14, and 12 from 20 nonsynonymous amino acid substitutions were identified in the E2, E4, and E6 genes respectively.

This study has provided one of the most comprehensive analyses of sequence variation within HPV 16 cancer isolates and the first HPV 16 sequence data from Australia and the South Pacific region. It has also provided evidence of new strategies by which HPV 16 may achieve malignant conversion without integration.