Lionel Crawford

An immunochemical investigation of SV40 T-antigens 2. Quantitation of antigens and antibody activities

Abstract The interaction of the two major forms of simian virus 40 (SV40) T-antigen, large-T and small-t, with antisera has been studied using immunoprecipitation followed by adsorption on to fixed Staphylococcus aureus Cowan 1. With sera derived from hamster bearing bearing tumors of SV40-transformed cells, the amounts of serum required for optimum precipitation of the two antigens were markedly different. Small-t required 15 to 30 times more serum than large-T. This effect can lead to underestimation of small-t or even failure to detect this species. The amount of small-t synthesized in SV40-infected CV-1 cells at late times after infection is substantial, more than equimolar with respect to large-T. Specific antiserum against the large-T polypeptide also precipitates small-t and the two antigens are immunoprecipitated coordinately, consistent with their sharing antigenic determinants. The only antiserum tested which failed to react with small-t was anti-U serum. Even in a sensitive radioimmunoassay, using the denatured polypeptide purified by gel electrophoresis as probe, anti-U serum had no detectable activity against small-t. Coordinate precipitation of large-T and a 53,000-dalton protein from extracts of SV40-transformed mouse cells did not appear to be due to their having shared antigenic determinants, but rather to the existence of a complex of large-T with the 53K protein. The latter protein appears to be a host protein, unrelated to large-T but complexed with it.

An immune complex assay for SV40 T antigen.

Abstract An assay is described for the detection and quantitation of T antigen, a protein present in cells transformed or infected by simian virus 40. In principle it is a direct immunoprecipitation of the antigen with serum from tumor bearing hamsters, followed by collection of the immune complexes formed on glass fiber filters. The complexed immunoglobulin is then exposed to 125I-labeled protein A, a protein with affinity for the Fc region of many immunoglobulins. The amount of protein A bound is then related to the amount of immunoglobulin and thus of antigen on the filter. With the appropriate sera this assay can probably be used for many other large antigens of biological interest.

The complex between simian virus 40 T antigen and a specific host protein

The small DNA tumour virus simian virus 40 (SV40) expresses only two proteins in the cells that it has transformed, large-T and small-t. Large-T has an apparent molecular mass of 94000 and genetic evidence indicates that it is necessary both for the induction and maintenance of the transformed state. In the lytic cycle of the virus the large-T protein acts to initiate viral DNA replication and appears to regulate its own transcription. These properties imply that the protein must interact with existing host cell mechanisms in a specific way and that an understanding of these interactions may be important in discovering how the protein acts in ‘transforming’ non-permissive cells. To identify these interaction structures in the host cell an immunochemical approach has been adopted, and has shown that a fraction of the large-T protein in transformed cells exists in a specific complex with a host-coded phosphoprotein. The host protein has an apparent molecular mass of 53000. The physiological role of this host protein-T antigen complex has been examined by studying the nature of the complex in a wide variety of different SV40 transformed cell lines. Although in SV40-transformed cells of rodent origin all the host protein present in the cell is complexed to large-T, it was found that in human cells transformed by SV40 only a fraction of the host protein is present in a complex and these cells contain large-T and the host protein in both the free and the complexed state. In SV40-transformed mouse cells that have been selected for loss of the transformed phenotype the complex was still present and in apparently normal amounts. This would indicate that no simple causal relation exists between the presence of the complex and the phenotype of the cell containing it.

Analysis of human papillomavirus type 16 polypeptides in transformed primary cells

The close association between human papillomavirus type 16 and cervical cancer implies some role for the virus in the development of this cancer. It has been demonstrated that HPV-16 DNA sequences in the presence of activated ras are capable of transforming primary baby rat kidney cells. This communication describes the characterization of these transformed cells. All cell lines are shown to be tumorigenic in immunocompetent rats and there is continued expression of the HPV-16 E6 and E7 polypeptides in a number of the cell lines analyzed, suggesting a role for at least one of these proteins in the maintenance of the transformed phenotype.