Cellular tumor antigen p53 (153-165)

SV40 mutants bearing either amino acid substitution or in-frame deletion/insertion mutations in a region of the gene for large T antigen encoding a stretch of hydrophobic residues were analyzed for their behavior in permissive and nonpermissive cells. One of the mutants, with an Ile(573)-Phe substitution had a phenotype indistinguishable from that of wild-type SV40. The remaining three mutants were not viable and were defective for DNA replication. In addition, they displayed a cell-type specificity with respect to transformation; namely, they transformed the mouse C3H10T1/2 cell line, although with a reduced efficiency relative to wild-type, but were unable to transform the rat REF52 cell line. None of the T antigens from the defective mutants formed a complex with the cellular protein p53, indicating that the T-antigen-p53 complex is not required for the transformation of C3H10T1/2 cells.

Simian virus 40 (SV40) mutant 5002 carries base pair substitutions of C-5109----T and C-5082----T. These mutations lie in a region of the genome that encodes amino acids common to the large and small viral tumor antigens (T and t antigens, respectively) and result in amino acid substitutions of Leu-19----Phe and Pro-28----Ser. In contrast to wild-type SV40, which produces large plaques that are clearly visible 8 days postinfection, mutant 5002 is defective for productive infection, producing tiny plaques that arise at around 21 days postinfection. However, 5002 is capable of replicating viral DNA and producing normal amounts of capsid proteins, indicating that the mutations alter an activity of T antigen that is required subsequent to DNA synthesis, such as maturation, viral assembly, or release of virions. The mutant T antigen has normal ATPase activity, is phosphorylated in a manner that is indistinguishable from that of the wild-type T antigen, and retains the ability to oligomerize. 5002 complements mutants defective in T antigen host range-adenovirus helper function for productive infection. Thus, T antigen encodes two activities that affect at least two different steps in viral infection other than DNA replication, one inactivated by mutations in the host range-adenovirus helper domain and one inactivated by the mutations present in 5002. The 5002-encoded T antigen is also defective for transformation of REF52 cells when expressed from the normal SV40 early promoter, although this defect can be partially overcome by expressing the protein from stronger promoters.

Two sets of mutations were introduced into a region of the JC virus (JCV) large tumor (T) antigen involved in binding the retinoblastoma susceptibility gene product (Rb). The first set converted the JCV sequences to those found in the corresponding region of the simian virus 40 (SV40) T antigen. The second set contained sequence changes predicted to abolish Rb binding. Each of these mutations was also inserted into a chimeric T antigen (MSTn) composed of JCV and SV40 sequences at its amino- and carboxy termini, respectively. The JCV T antigen is less efficient than its SV40 counterpart at transforming Rat2 cells and at binding Rb and viral DNA. These activities were altered in the two sets of mutants generated in this study. A JCV T antigen mutant having an SV40-like Rb-binding domain exhibited increased DNA binding activity while, unexpectedly, displaying decreased Rb binding and wild-type transforming behavior. A mutant T antigen that was unable to bind Rb exhibited decreased DNA binding and failed to transform Rat2 cells. Both mutants were defective for DNA replication and did not produce infectious virions. Additional phenotypic changes were observed when each mutation was introduced into the chimeric MSTn T antigen. As the oligomerization state of SV40 T antigen is known to influence several of its activities, including Rb binding, the quaternary structure of the T proteins used in this study was assessed by sucrose gradient sedimentation. The SV40 and chimeric MSTn T antigen sedimented as a mixture of monomers/dimers and higher oligomers, whereas the JCV T antigen sedimented predominantly as monomers/dimers; neither mutation in the T antigen Rb-binding motif affected the sedimentation profiles of the parental T proteins. Restricted biochemical activity of the JCV T protein relative to that of SV40 supports the suggestion that this regulatory protein contributes to the attenuation of the JCV lytic cycle.