The large T antigen encoded by simian virus 40 (SV40) plays essential roles in the infection of permissive cells, leading to production of progeny virions, and in the infection of nonpermissive cells, leading to malignant transformation. Primary mouse embryo fibroblasts (MEFs) are nonpermissive for SV40, and infection by wild-type SV40 leads to immortalization and transformation of a small percentage of infected cells. We examined the ability of an extensive set of mutants whose lesions affect SV40 large T antigen to immortalize MEFs. We found that immortalization activity was retained by all mutants whose lesions are located upstream of codon 346. This includes a mutant lacking amino acids 168 to 346. We previously showed (M. J. Tevethia,

Mouse C3H 1OT1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 1OT1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 1OT1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.

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We have developed five mouse monoclonal antiidiotypic antibodies to the 35/56 (Ab1) rat monoclonal that neutralizes retroviral infectivity by binding to the gp70f epitope of murine leukemia retrovirus. The anti-Id nature of these five Ab2s was evidenced by their inability to react with a panel of six other rat IgG2a kappa monoclonals isotype-matched to the 35/56 anti-gp70f mAb1, including two to the distinct epitopes "g" and "h" of gp70, or to normal rat IgG2a. On the basis of several competition assays four mAb2 were clearly either directed to the paratope of anti-gp70f mAb1 (.1C7, .1B, and .E) or not (.A, representing a noninternal image Ab2 alpha anti-Id). The P3E8 mAb2 was difficult to classify. Based on relative efficiency in these assays, .1C7 was chosen for further study, and upon injection was able to induce Ab3 responses in C57BL/6, BALB/c, and CBA mice. The fact that the Ab3 activity was detected in a competitive ELISA in which the hyperimmune antisera blocked the binding of Ab1 to Ab2, plus the ability to raise Ab3 neutralizing antibodies in three different mouse strains were consistent with .1C7 as an internal image Ab2 beta anti-Id. These results thus indicate the potential for internal-image monoclonal antiidiotypic antibody-based vaccines for retroviral diseases.