Human papillomavirus (HPV) type 16 and 33 E6/E7 region transcripts in tonsillar carcinomas can originate from integrated and episomal HPV DNA.

This study was undertaken to determine whether human papillomavirus (HPV) E6/E7 gene transcription in tonsillar carcinomas is correlated with viral DNA integration. Therefore, tonsillar carcinomas containing HPV-16 (n = 2) and HPV-33 (n = 2) DNA were analysed for the viral physical state and transcription of the E6/E7 region. Southern blot analysis, DNA polymerase chain reaction (PCR) and, eventually, two-dimensional gel electrophoresis revealed indications for the presence of only episomal DNA in the HPV-16-containing biopsies and only integrated DNA in one HPV-33-containing biopsy. The second HPV-33-containing carcinoma, from which one biopsy and two resected tumour specimens were analysed, showed a rather complex physical state profile. The biopsy of this tumour contained only episomal DNA, one resected tumour part contained only integrated DNA and the remaining tumour part contained both integrated and episomal HPV-33 DNA. Independent of the viral physical state, all biopsies and resected tumour parts tested showed the presence of E6/E7 transcripts as determined by RNA PCR. The results indicate that E6/E7 transcripts in tonsillar carcinomas can originate from integrated as well as episomal HPV DNA.

Prevalence and expression of human papillomavirus in tonsillar carcinomas, indicating a possible viral etiology.

The presence of human papillomavirus (HPV) DNA was assessed in biopsies of tonsillar carcinomas (n = 10) and cases of tonsillitis (n = 7), serving as controls, by general-primer-mediated PCR (GP-PCR). All carcinomas appeared HPV-positive, whereas all cases of tonsillitis were HPV-negative. Additional type-specific PCR for HPV 6, 11, 16, 18, 31 and 33 revealed that 4 carcinomas contained HPV 16 DNA, 4 contained HPV 33 DNA and 1 contained an HPV 16/33 double infection. False positivity was excluded by additional Southern blot analysis of type-specific PCR-positive samples (n = 4). Further characterization of GP-PCR products by sequence analysis revealed that 2 carcinomas contained still uncharacterized HPV genotypes; one of these also contained HPV 33 DNA and one was negative by type-specific PCR. Application of RNA PCR revealed expression of HPV 16 or HPV 33 E7 encoding spliced E6*1 transcripts in all tonsillar carcinomas (n = 4) examined. Additional non-radioactive RNA in situ hybridization performed on 3 biopsies revealed the presence of HPV 16 or HPV 33 E7 transcripts exclusively localized within the carcinoma cells, whereas stroma stained negative. These findings strongly support a role for certain HPV types in the pathogenesis of tonsillar carcinomas.

General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions.

We recently described the detection of potentially novel human papillomaviruses (HPV) genotypes (HPV types X [HPV X]) in cervical smears (A. J. C. van den Brule, C. J. L. M. Meijer, V. Bakels, P. Kenemans, and J. M. M. Walboomers, J. Clin. Microbiol. 28:2739-2743, 1990) by using the general primer-mediated polymerase chain reaction method (GP-PCR). In this study, the HPV specificities of GP-PCR products were determined by sequence analyses. M13 bacteriophage clones of PCR products derived from cloned unsequenced HPV genotypes 13, 32, 35, 43, 44, 45, 51, and 56 were subjected to dideoxy sequencing. Analyses of the putative amino acid sequences of these HPV types in addition to published HPV sequence data revealed stretches of highly conserved amino acid residues present in all HPV types, resulting in an HPV amino acid consensus sequence. Subsequently, HPV X-specific PCR products found in premalignant cervical lesions (n = 3), carcinomas in situ (n = 6), and invasive cancer (n = 6) were analyzed for their nucleotide sequences. Comparison of these sequences with published HPV nucleotide sequences and data obtained in this study revealed three HPV type 35, two HPV type 45, one HPV type 51, two HPV type 56, and six unique HPV X sequences, of which three types were present in four cases of carcinomas (in situ). The nucleotide sequences determined appeared to be unique after a data bank search. Furthermore, the sequences of all HPV X isolates matched the HPV amino acid consensus sequence, thus confirming HPV specificity. This study illustrates the power of GP-PCR in combination with sequence analysis to determine HPV specificity and genotyping of PCR products derived from sequenced as well as unsequenced HPVs, including novel, not yet identified HPV types.

Human papillomavirus type 33 in a tonsillar carcinoma generates its putative E7 mRNA via two E6* transcript species which are terminated at different early region poly(A) sites.

Human papillomavirus type 33 (HPV-33)-specific early region transcripts in a tonsillar carcinoma were analyzed by using the RNA polymerase chain reaction method. A total of five cDNA species including species with potential to encode E6*I, E6*II, and E6*III, could be identified. As determined by 3' cDNA end mapping, one E6*I cDNA species was found to utilize a novel early region poly(A) site and was polyadenylated at or near the putative initiation codon of the E1 open reading frame (ORF). Compared with the HPV-16 and HPV-18 E6* mRNAs, the HPV-33 E6*I and E6*II species utilize different splice acceptor sites, the latter being localized within the E7 ORF. Furthermore, HPV-33 E6* mRNAs were found to contain a short overlapping ORF resulting in alternative coding potentials if translation were to start at an internal AUG codon within the E6 region. These results indicate that like HPV-16 and HPV-18, HPV-33 generates E6* mRNAs which may serve as efficient mRNAs for E7. However, HPV-33 has the ability to generate its putative E7 mRNAs by the utilization of two early region poly(A) sites, which offers the possibility of expressing E7 in different ways.

The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes.

A novel polymerase chain reaction (PCR) method was developed that permits the detection of 11 different human papillomavirus (HPV) genotypes using two general primer sets. By computer-assisted sequence analysis, two pairs of general primers were selected from the conserved L1 open reading frame and tested in the PCR on a set of cloned HPV genotypes. Experimental analysis showed that up to three mismatches between primers and target DNA did not influence the efficiency of the assay. The use of these primers in the PCR enabled the detection of HPV genotypes HPV-1a, -6, -8, -11, -13, -16, -18, -30, -31, -32 and -33, and was also successfully applied to well characterized cervical carcinoma cell lines and clinical samples. For the HPV types tested sub-picogram amounts of cloned DNA could be detected after general primer-mediated PCR and subsequent hybridization. The specificity of the amplification products was confirmed by blot hybridization procedures and RsaI restriction enzyme digestion. The results indicate that this PCR method can be a powerful tool for identifying novel HPV genotypes in dysplasias and squamous cell carcinomas suspected of having an HPV aetiology.