Variants of the long control region and the E6 oncogene in European human papillomavirus type 16 isolates: implications for cervical disease

Abstract

High-risk human papillomavirus types, especially type 16, are risk factors for cervical cancer. Preliminary studies suggest that HPV16 polymorphisms in the long control region or in the E6 gene may alter the oncogenic potential of the virus. This could partially explain why some lesions progress to cancer while others do not. A systematic study combining the long control region and E6 has not been undertaken. This prompted us to investigate the long control region and the E6 in northern European women infected with human papillomavirus 16. We identified the sequence variations of both regions and investigated the long control region promoter activity among various isolates. In addition, we correlated the distribution of long control region and E6 polymorphisms with disease status. We analyzed 45 samples from Swedish and Finnish women. The long control region and the E6 gene were sequenced after polymerase chain reaction long control region fragments of six European isolates covering the majority of polymorphisms in this region were ligated into the pALuc vector and used for luciferase assays. In European HPV16 isolates, polymorphisms in the long control region are more frequent than in the E6 gene. Nevertheless, the promoter function was slightly increased in only one of the tested European long control region variants. In addition, we found a specific European E6 variant, L83V, to be enriched in high-grade lesions and cancer rather than a specific European long control region variant. The difference in oncogenicity between European HPV16 genotypes is more probably due to an altered property of the corresponding E6 proteins rather than to an altered activity of the P97 promoter.

Figure 1

Sequence alterations relative to the LCR and E6 open reading frame of reference HPV16. Differences between the isolates and the HPV16 reference sequence (bold) are shown. The type of lesion from which the isolates are derived is indicated. Dashes indicate nucleotides identical to the reference sequence. Capital letters indicate alterations in the E6 open reading frame that result in amino acid changes, while lower case letters indicate silent mutations. The position of amino acid change is indicated numerically. The letter preceding this number refers to the prototype amino acid, and the letter following it refers to its substitution. The polymorphisms not reported previously appear in italics. Swedish cases are denoted ‘US’ and Finnish cases ‘F’ or ‘T’. HPV=human papillomavirus; LCR=long control region; ORF=open reading frame; R=reference sequence (Seedorf et al, 1985); L/HCIN=low-grade/high-grade cervical intraepithelial neoplasia.

Figure 2

Luciferase expression under the control of the HPV16 LCR-fragments of the different European isolates The results for the isolates US53, 50, 17, 134, 56 and 58 as well as the mutated LCR m-US56 are shown. The mean values of six independent assays together with their standard deviations are demonstrated. The LCR of prototype HPV16 was used as a reference clone and set to the value 1.0 in all assays.