Transcriptional activity of human papillomavirus type 6 in respiratory tract papillomata

We have investigated the molecular basis for differences that we observed in the biological activities of genetically related but distinguishable human papillomavirus type 6 (HPV-6) subtypes. To analyse tissue-specific differences in replication and transcription, and to identify viral gene products important in the benign transformation of epithelial cells, we have modified procedures utilizing guanidinium isothiocyanate and density gradient centrifugation to facilitate the extraction of relatively undegraded DNA and RNA from 21 biopsy specimens of respiratory tract papillomata. Southern transfer analysis was used to characterize the viral genome, and to demonstrate that relative quantities of viral DNA in lesions varied but that the range was similar in lesions induced by HPV-6c, -6d, -6e and -6f. Dot blot analysis of the amount of viral RNA in comparison with the amount of 28S ribosomal RNA indicated that the relative level of viral RNA in each lesion varied considerably and that on average there was approximately twice as much viral RNA in HPV-6c-induced lesions as in HPV-6d-, -6e- or -6f-induced lesions. In dot blot and Northern analyses, hybridization of RNA from HPV-6c-induced lesions with HPV-6c DNA gave a stronger signal than hybridization of the same RNA with an HPV-6e probe, and vice versa. These differences in hybridization intensities with subtype-specific probes indicate that the most abundant RNA species are transcribed from parts of the genome that show sequence divergence between these two subtypes. Northern analysis demonstrated the predominant viral transcript to be about 1200 nucleotides in length in lesions induced by each of the four subtypes.