Authentic simian virus 40 (SV40) has been detected in association with human choroid plexus and ependymoma tumors, and SV40‐like DNA sequences have been found in some human osteosarcomas. We report here an analysis of human osteosarcoma samples for the presence of SV40 DNA using PCR and primers directed at 4 distinct sites of the SV40 genome, coupled with sequence analysis. Authentic SV40 DNA sequences were detected in 5 of 10 osteosarcoma tumor samples. The SV40 regulatory region in each case was identical and of archetypal length (non‐duplicated enhancer), as is usually found in natural isolates of SV40 from monkeys and in human brain tumors. A section of the gene that encodes a viral late gene product (VP1) was detected in 5 of 10 tumors and had an exact match with the known sequence of SV40. Two separated segments of the large T‐antigen (T‐ag) gene were found in the same 5 tumors. Analysis of the DNA sequences encoding the T‐ag carboxy terminus revealed sequence variation among the tumors, as observed previously in viral DNA associated with human brain tumors. There does not appear to be a preferential association of a T‐ag variable domain sequence with a given tumor type. No sequences from the regulatory region of human polyomaviruses JCV and BKV were detected in the bone tumors. We also noted less efficient recovery of SV40 DNA from tumor samples fixed in paraffin as compared to frozen tumors. Our results confirm the presence of SV40 DNA in human bone tumors and, based on the sequence variation observed for the carboxy terminus of the T‐ag gene, suggest that there is not a specific SV40 strain associated with human osteosarcomas. Int. J. Cancer 72:791–800, 1997. © 1997 Wiley‐Liss, Inc.

Most of the simian virus 40 (SV40) genome is conserved among isolates, but the noncoding regulatory region and the genomic region encoding the large T-antigen C terminus (T-ag-C) may exhibit considerable variation. We demonstrate here that SV40 isolates differ in their oncogenic potentials in Syrian golden hamsters. Experimental animals were inoculated intraperitoneally with 10 7 PFU of parental or recombinant SV40 viruses and were observed for 12 months to identify genetic determinants of oncogenicity. The viral regulatory region was found to exert a statistically significant influence on tumor incidence, whereas the T-ag-C played a minor role. Viruses with a single enhancer (1E) were more oncogenic than those with a two-enhancer (2E) structure. Rearrangements in the 1E viral regulatory region were detected in 4 of 60 (6.7%) tumors. Viral loads in tumors varied, with a median of 5.4 SV40 genome copies per cell. Infectious SV40 was rescued from 15 of 37 (40%) cell lines established from tumors. Most hamsters with tumors and many without tumors produced antibodies to T antigen. All viruses displayed similar transforming frequencies in vitro, suggesting that differences in oncogenic potential in vivo were due to host responses to viral infection. This study shows that SV40 strains differ in their biological properties, suggests that SV40 replicates to some level in hamsters, and indicates that the outcome of an SV40 infection may depend on the viral strain present.Simian virus 40 (SV40) is a member of the family Polyomaviridae and is known for its ability to induce malignancies in the Syrian golden hamster (Mesocricetus auratus) model (9-11, 16, 20, 32). SV40 was discovered as an inadvertent contaminant of early forms of poliovirus and adenovirus vaccines (9, 46) that were prepared in primary cultures of kidney cells from rhesus monkeys, which are often naturally infected with the virus (9, 43, 51). Since its discovery, SV40 has been an important model for studies of virus-induced cancers and of viral effects on eukaryotic cell processes (1, 5). SV40 has been found to cause human infections and to be associated with some human malignancies (9, 21, 52).Phylogenetic analysis has recently established that strains of SV40 exist and can be grouped into clades or genogroups (18). Strains are identified by nucleotide differences at the C terminus of the large tumor antigen (T-ag) gene (T-ag-C) that result in amino acid changes in the protein. The SV40 large T-ag protein is essential for viral replication and is the major viral oncoprotein (1, 9, 32). Strains of SV40 can diverge in the structures of their noncoding regulatory regions (9,23,26,45), generating what are termed variants. SV40 variants containing a partial or complete duplication of the 72-bp enhancer element or other sequence rearrangements are designated as having complex regulatory regions, and those with one enhancer are designated as having a simple or archetypal regulatory region structure (23,26). It has been demonstrated that increased numbers of enha...

In adenovirus-infected cells, the epidermal growth factor receptor (EGF-R) is internalized from the cell surface via endosomes and is degraded, and the E3 10,400-dalton protein (10.4K protein) is required for this effect (C. R. Carlin, A. E. Tollefson, H. A. Brady, B. L. Hoffman, and W. S. M. Wold, Cell 57:135-144, 1989). We now report that both the E3 10.4K and E3 14.5K proteins are required for this down-regulation of EGF-R in adenovirus-infected cells. Down-regulation of cell surface EGF-R was demonstrated by results from several methods, namely the absence of EGF-R autophosphorylation in an immune complex kinase assay, the inability to iodinate EGF-R on the cell surface, the formation of endosomes containing EGF-R as detected by immunofluorescence, and the degradation of the metabolically [35S]Met-labeled fully processed 170K species of EGF-R. No effect on the initial synthesis of EGF-R was observed. This down-regulation was ascribed to the 10.4K and 14.5K proteins through the analysis of cells infected with rec700 (wild-type), dl748 (10.4K-, 14.5K+), or dl764 (10.4K+, 14.5K-) or coinfected with dl748 plus dl764. Further evidence that the 10.4K and 14.5K proteins function in concert was obtained by demonstrating that the 10.4K protein was coimmunoprecipitated with the 14.5K protein by using three different antisera to the 14.5K protein, strongly implying that the 10.4K and 14.5K proteins exist as a complex. Together, these results indicate that the 10.4K and 14.5K proteins function as a complex to stimulate endosome-mediated internalization and degradation of EGF-R in adenovirus-infected cells.

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The entire early regions of three human isolates of simian virus 40 (SV40), as well as two laboratory strains recovered from monkeys, were sequenced. The early coding region of each isolate contains a number of nucleotide differences when compared to the reference strain SV40-776. These differences result in some changes in the predicted amino acid sequence of the unique region of small t-antigen and in the carboxy (C) terminus of large T-antigen. The amino acid sequence of the remainder of large T-antigen was absolutely conserved among all isolates. Thus, SV40 large T-antigen contains a variable domain at the C-terminal end of the molecule.