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Analysis of a large-T-antigen variant expressed in simian virus 40-transformed mouse cell line mKS-A
1Fakultät für Biologie, Universität Konstanz, Federal Republic of Germany.
Abstract:
Earlier reports had suggested that the large T antigen expressed in simian virus 40 (SV40)-transformed mKS-A cells may be replication defective. Our experiments support these earlier observations showing that the mKS-A T antigen has a reduced DNA-unwinding activity in vitro. To investigate the molecular basis for this defect, we have isolated from an mKS-A genomic library an EMBL-3 bacteriophage clone carrying in its insert a full-length SV40 DNA element that most likely encodes the expressed T-antigen variant. DNA sequencing revealed only one nonconservative amino acid exchange, Asp to Asn at residue 636. Surprisingly, when a plasmid clone carrying the mKS-A T-antigen-coding sequence was transfected into monkey cells, we found that it replicated quite efficiently, probably suggesting that a high nuclear concentration of the variant T-antigen form compensates for the partial biochemical defect. However, a high nuclear concentration of T antigen was also found in mKS-A T-antigen-transformed mouse cells, yet a fusion of these cells to permissive monkey cells failed to induce in situ replication and excision of integrated SV40 DNA. We discuss possible reasons for the different behavior of T antigen in monkey cells and in mouse cells and suggest that one possibility for the replication-negative phenotype in transformed cells may be related to the fact that T antigen forms a tight complex with the cellular p53 protein in mouse cells but not in monkey cells.
Insights
Simian virus 40 (SV40) T-antigen variants can exhibit reduced DNA unwinding activity. This defect is overcome in monkey cells by high concentrations, but not in mouse cells due to p53 complex formation.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Simian virus 40 (SV40) large T-antigen is crucial for viral replication.
- Previous studies indicated potential replication defects in SV40 T-antigen variants expressed in mKS-A cells.
Purpose of the Study:
- To investigate the molecular basis of the replication defect in SV40 T-antigen from mKS-A cells.
- To determine the role of specific mutations and cellular context in T-antigen function.
Main Methods:
- Isolation and sequencing of SV40 DNA from mKS-A cells.
- Transfection of SV40 T-antigen constructs into monkey cells.
- Cell fusion experiments between SV40-transformed mouse and monkey cells.
- Analysis of T-antigen DNA-unwinding activity in vitro.
Main Results:
- The mKS-A T-antigen variant exhibits reduced in vitro DNA-unwinding activity due to a single amino acid change (Asp636Asn).
- Transfection into monkey cells showed efficient replication, suggesting compensation by high nuclear T-antigen concentration.
- SV40-transformed mouse cells, despite high T-antigen levels, failed to replicate SV40 DNA upon fusion with monkey cells.
Conclusions:
- The SV40 T-antigen variant's replication efficiency is cell-type dependent.
- High nuclear concentration can compensate for biochemical defects in monkey cells.
- Differential interaction of T-antigen with cellular p53 protein in mouse versus monkey cells may explain the replication defect in transformed mouse cells.