On the functional roles of simian virus 40 large and small T-antigen in the induction of a mitotic host response.

The early gene of wild-type (wt) SV40 specifies two related proteins, referred to as large (Mr 88,000) and small (Mr 19,000) T-antigen. Infection with wt SV40 of Go/G1-arrested monkey kidney and CV-1 cell cultures induced in virtually 100% of the cells T-antigen synthesis, followed by a mitotic reaction and the production of SV40 DNA. Parallel cultures were infected with SV40 deletion mutants that produce either no small T-antigen (d1883) or only trace amounts of a truncated form (d1891). Kinetics of synthesis and accumulation of large T-antigen was closely similar to that observed with wtSV40 whereas apparently only 50-60% of the cells participated in the mitotic reaction and the production of viral DNA. These results and those obtained from a comparative study on the abortive (transforming) infection in Go-arrested mouse tissue culture cells indicate that synthesis of large T-antigen alone is sufficient to trigger in 50-60% of the infected cells a mitotic reaction.     

Insensitivity of a ricin-resistant mutant of Chinese hamster ovary cells to fusion induced by Newcastle disease virus.

The simian virus 40 (sv40) tumor antigen (T-antigen) and tumor-specific transplantation antigen (TSTA) have been partially purified and studied to clarify their relationship. The T-antigen and the TSTA were partially purified from nuclei of SV AL/N cells, and SV40-transformed mouse embryo fibroblast line, by precipitation with ammonium sulfate and chromatography on DEAE- and DNA-cellulose. The T-antigen was assayed by complement fixation, and the TSTA was assayed by its ability to immunize mice against SV40-containing ascites tumor cells. When T-antigen- and TSTA-containing preparations were sedimented through sucrose gradients, each antigen had a major peak of activity at a sedimentation coefficient of 6.7 and minor peaks in other regions. Antiserum against T-antigen (from tumor-bearing hamsters) immunoprecipitated the TSTA activity. A preparation of T-antigen from human SV80 cells, which exhibited only one protein band after sodium dodecylsulfate-polyacrylamide gel electrophoresis, had TSTA activity when as little as 0.6 microgram of protein per mouse was used for immunization. These experiments demonstrate that the T-antigen, the product of the SV40 early A gene is capable of inducing specific immunity against transplantation of SV40-transformed tumor cells in mice.     

Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells.

The 54K cellular tumor antigen has been translated in vitro, using messenger ribonucleic acids from simian virus 40 (SV40)-transformed cells or 3T3 cells. The in vitro 54K product could be immunoprecipitated with SV40 tumor serum and had a peptide map that was similar, but not identical, to the in vivo product. The levels of this 54K protein in SV3T3 cells were significantly higher than those detected in 3T3 cells (D. I. H. Linzer, W. Maltzman, and A. J. Levine, Virology 98:308-318, 1979). In spite of this, the levels of translatable 54K messenger ribonucleic acid from 3T3 and SV3T3 cells were roughly equivalent or often greater in 3T3 cells. Pulse-chase experiments with the 54K protein from 3T3 or SV3T3 cells demonstrated that this protein, once synthesized, was rapidly degraded in 3T3 cells but was extremely stable in SV3T3 cells. Similarly, in an SV40 tsA-transformed cell line, temperature sensitive for the SV40 T-antigen, the 54K protein was rapidly turned over at the nonpermissive temperature and stable at the permissive temperature, whereas the levels of translatable 54K messenger ribonucleic acid at each temperature were roughly equal. These results demonstrate a post-translational regulation of the 54K cellular tumor antigen and suggest that this control is mediated by the SV40 large T-antigen.     

pZ402, an improved SV40-based shuttle vector containing a T-antigen mutant unable to interact with wild-type p53

Shuttle vectors are useful tools for studying DNA replication and mutagenesis. SV40-based shuttle vectors are popular because of their ease of use and quick results. However, one complication with the use of SV40-based shuttle vectors is the interaction of cellular p53 protein with the T-antigen of SV40. Wild-type, but not mutant, p53 has been shown to be involved in DNA replication and DNA repair. To address this concern, we have modified an SV40-based shuttle vector, pZ189, by exchanging the wt T-antigen for a mutant SV40 T-antigen, which is unable to bind with p53. This shuttle vector, pZ402, provides us with a tool to study DNA replication and genomic instability in cells with varying genetic backgrounds without interference from the interaction of T-antigen with p53.     

SV40 large T-antigen and transformation related protein p53 are associated in situ with nuclear RNP structures containing hnRNA of transformed cells

The localization of SV40 large T-antigen (T-Ag) and the cellular protein p53 in the nuclei of mouse and human SV40-transformed cells and of a methylcholanthrene-transformed mouse cell line, was studied. Their detection by ultrastructural immunocytochemistry with specific monoclonal antibodies employed two complementary methods used in parallel. These consisted of indirect immunoperoxidase labelling carried out before embedment on Triton-permeabilized cells, or indirect immunogold labelling applied to thin sections of cells embedded in Lowicryl K4M. The results indicate that in SV40-transformed cells both proteins are chiefly localized on peri- and interchromatin RNP fibrils. This shows that they occur in structures involved in the synthesis and processing of hnRNA. The nucleoli and chromatin did not appear to be labelled. In methylcholanthrene-transformed cells the protein p53 (in the absence of large T-Ag) was also detected on peri- and interchomatin fibrils. Taken together with recent results which demonstrated that, during lytic infection, T-Ag was associated chiefly with cellular chromatin (Harper, F, Florentin, Y & Puvion, E, Exp cell res 161 (1985) 434) [33], our experiments provide evidence that the transforming function of SV40 large T-Ag is dissociable from its function in SV40 lytic infection in terms of its subnuclear distribution.