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In animal studies, polyoma viruses have been found to be viral agents for oncogenesis and to produce a wide range of pathological lesions in experimental animals, including a variety of neoplastic tumors. The human polyoma viruses (JCV and BKV), along with their simian cousin (SV40), are ubiquitous viruses that are primarily associated with progressive multifocal leukoencephalolopathy (PML) and hemorrhagic cystitis, respectively, under specific conditions in immunocompromized individuals. Currently, polyoma viruses are now undergoing increasing scrutiny as possible causes for several human cancers. Evidence has been mounting recently that JCV, BKV as well as SV40 are potential oncogenic viruses in humans as well.

To explore the molecular mechanisms by which retinoic acid inhibits oncogenic transformation, we have examined the effects of retinoic acid on the polyomavirus-induced transformation of rat fibroblasts. Treatment of rat F111 fibroblasts with high concentrations of retinoic acid (10(-6) M) partially inhibited the ability of polyomavirus to induce dense focus formation (50-70%). This effect was not secondary to a retinoic acid-dependent block of cellular proliferation. To address the role of the retinoic acid receptor (RAR-alpha) in mediating the transformation-inhibitory effect of retinoic acid, we have overexpressed either RAR-alpha or cellular retinoic acid-binding protein I (CRABP) cDNAs in F111 cells. Introduction of a CRABP I expression vector did not alter the responsiveness of F111 cells to retinoic acid in any detectable fashion. In contrast, overexpression of RAR-alpha increased the sensitivity of F111 cells to the transformation-inhibitory action of retinoic acid by 10- to 100-fold. At high concentrations, retinoic acid inhibited transformation of F111-RAR cells by polyomavirus by about 90%. At near physiological concentrations, retinoic acid inhibited transformation by 25-50% in F111-RAR cells but not in control cells. Retinoic acid did not inhibit either the synthesis of polyoma middle T (mT) or pp60c-src, the cellular target for mT action, or the formation of mT:pp60c-src:PI-3 kinase (phosphatidylinositol-3 kinase) complexes. Therefore, RAR-alpha was not acting as a negative regulator of expression of either the polyomavirus middle T oncogene or the cellular proto-oncogene, c-src. It seems likely that RAR-alpha regulates the expression of cellular genes whose products interact in some way with mT-regulated signaling pathways, leading to a ligand-dependent suppression of polyoma transformation. In addition, RAR-alpha overexpression selectively inhibits the serum-stimulated expression of the c-fos gene, but does not affect the expression of a number of other serum- and polyomavirus-inducible genes including c-jun, junB, c-myc and actin.

By testing recombinants between "high tumor" (inducing a high incidence of tumors) and "low tumor" (inducing a low incidence of tumors) strains of polyomavirus, we have previously shown that the key determinant(s) for induction of a high tumor profile resides in coding regions of the high tumor strain (R. Freund, G. Mandel, G. G. Carmichael, J. P. Barncastle, C. J. Dawe, and T. L. Benjamin, J. Virol. 61:2232-2239, 1987). Three single-amino-acid differences between the PTA (high tumor) and RA (low tumor) virus strains have now been identified by DNA sequencing, one each in the large T antigen, in the region common to the middle and small T antigens, and in the major capsid protein VP1. Further tests of appropriate recombinants and oligonucleotide-induced mutants show that VP1 of PTA is the major determinant for induction of a high tumor profile, including all tumors of epithelial origin. The differential effect of the VP1s of PTA and RA on the tumor profile is discussed in terms of a likely contribution of the polymorphic region of VP1 to binding of receptors and infection of different cell types in the animal. The polymorphism in the large T antigen has a more restricted action, which is seen only when tested in virus carrying the VP1 type of PTA; the PTA large T antigen then promotes more rapid growth of tumors of salivary gland and thymus than the RA large T antigen.

The human papovavirus, BK, appeared weakly oncogenic in newborn hamsters and was able to induce in vitro transformation of rat kidney cells. The infectivity of BK virus DNA was determined by employing the DEAE-dextran method. In human embryonic cells the infectivity was approx. 10(5) p.f.u./mug of DNA. The transforming ability of BK virus in primary rat kidney cells was measured by employing the calcium method and appeared to vary from 1 to 10 foci per mug of DNA.

The transforming activity of polyoma virus was verified in vitro in organ cultures of 12-day old rat visceral yolk sacs and embryos. Organ cultures of 18-day old fetal organs were also included. The transforming capacity of the virus was found to be restricted to the endothelial cells of the rat visceral yolk sac. The neoplastic endothelial cells are readily transplantable and possess the polyoma tumor-specific transplantation antigens (TSTA).

The oncogenicity of the human polyomavirus BK (BKV) was tested in newborn inbred rats. It was found that the tumor rate was negatively correlated with the levels of T antibody 3 months after inoculation and the frequency of animals with detectable T antibodies 1.5 months after inoculation. By contrast, no influence of viral HI titers on the tumor rates was found. Thymectomy of animals resulted in most experiments in increased tumor rates. Inoculation with BKV of animals later than 24 hours after birth yielded a decrease of tumor rates. The results obtained suggest that T antibody titers present at a critical time after inoculation are associated with low oncogenicity of BKV. The oncogenicity of BKV was comparatively tested in rat strains possessing the allele "l" or the allele "a", respectively. The oncogenicity was significantly higher in rats with the allele "l" than in rats with the allele "a". Rats with the allele "l" showed lower T antibody response than rats with the allele "a". These differences could be explained by the finding that cells of "a" origin showed in vitro a higher percentage of T antigen bearing cells than did cells of a strain possessing the allele "l". In comparison to previous results obtained with BKV inoculated outbred WISTAR rats, the oncogenicity of comparable BKV doses in inbred rats was generally higher and the latency period of tumor manifestation shortened.

In an attempt to gain insight into the mechanism of oncogenic transformation by BK virus (BKV), a human papovavirus, we have probed for BKV sequences in transformed hamster cells in which oncogenic transformation had occurred as a result of transfection by human tumor DNA positive for BKV sequences. Even though the sources of the transfecting DNA contained BKV sequences, the transformed hamster cells which arose from the transfection for the most part did not retain BKV sequences. In only one barely detectable case was BKV-specific DNA found associated with chromosomal DNA, and in only a small minority of the transformed cells was BKV DNA detected in the Hirt supernatant, indicating an episomal configuration. Even in these few cases where BKV sequences were present in an episomal form, altered migration on gels of some BKV-positive bands (compared to bands derived from cloned viral DNA) suggested deletions and rearrangements of BKV DNA. We employed several different probe methodologies for these studies, including nick-translation, random primer and a non-isotopic biotinylated probe which gave a sensitivity that could detect better than 0.01 copy of viral genome per diploid cell. We conclude that transformation by transfection with human tumor DNA does not require persistence of the BKV viral genome, suggesting that either BKV virus was irrelevant to original oncogenesis, in analogy with models proposed by others for herpesvirus oncogenesis.

We have measured the quantitative binding of the radioactively labeled agglutinin (125)I-concanavalin A to normal mammalian cells and simian virus 40- and polyoma virus-transformed cells from tissue culture. Parallel measurements of the amount of (125)I-concanavalin A necessary to cause agglutination of the cells in suspension were carried out. The transformed and nontransformed cells used for these experiments show large differences in their ability to be agglutinated by (125)I-concanavalin A. However, these cell lines have the same number of specific binding sites and similar affinities for the agglutinin whether transformed, trypsinized, or nontransformed. We conclude that the differential capacity of concanavalin A to agglutinate transformed cells relative to normal cells does not result from differences in the number of binding sites between the two types of cells.

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.