VIRAL TRANSCRIPTS IN RABBIT (SHOPE) PAPILLOMA VIRUS INDUCED BENIGN AND MALIGNANT TUMORS: CHARACTERIZATION AND MAPPING OF TRANSCRIPTS

In the non-virus producing tumors induced by papilloma viruses, virus specific antigens have not been detected so far and the presence of virus specific transcripts has been established only in the Shope system. It was shown by single phase liquid hybridization that RNA of low abundance and homologous to 6 to 12% of the viral DNA was present in papillomas and carcinomas. To characterize these transcripts, a method for preliminary mapping was developed which did not depend on the isolation of intact RNA from tumors. The method involved the iodination of viral transcripts and their hybridization to DNA filters with restricted Shope DNA in excess. The low abundance of the viral transcripts required a preselection of these by hybridization to Shope DNA-cellulose and the subsequent rigorous removal of DNA leaking from the DNA cellulose. Although DNA contamination remained detectable, the method permitted to identify areas of the genome which were transcribed.; When a cell line derived from the Shope virus induced transplantable VX-7 carcinoma became available, it appeared feasible to isolate intact virual RNA. This transplantable tumor as well as the derived cell line both contained transcripts of low abundance and homologous to 11% of the viral DNA. Extraction of RNA with guanidine hydrochloride permitted the isolation of intact RNA from whole cells, however, at a low yield. The introduction of a sonication step improved the RNA recovery greatly and two major viral transcripts of 1.3 and 2.0 kb could be identified. Both transcripts were present in the cytoplasm and it was shown indirectly that both were also polysome associated. Two minor larger transcripts of 3.5 and 4.8 kb appeared to be restricted to the nucleus. The two major transcripts were mapped by hybridization of RNA transfer blots to subgenomic probes and both were shown to be spliced and colinear.; Finally, the modified guanidine hydrochloride extraction procedure permitted the isolation of intact RNA from both benign and malignant tumors. All these tumors contained the 1.3 and 2.0 kb major and the 3.5 and 4.8 kb minor transcripts. A difference noted between carcinoma and papilloma was that the 1.3 kb transcript is more prominent than the 2.0 kb transcript in carcinomas while the opposite was found in a papilloma. The presence of a 1.3 and 2.0 kb virus specific transcript in all non-virus producing tumors induced by the Shope virus and particularly its presence in a transplantable tumor established over 30 years ago, suggested that their translation products are essential for the maintenance of the neoplastic state.