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DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication
Abstract:
Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.
Insights
Simian virus 40 (SV40) T-antigen variants were studied for their DNA binding and replication abilities. A single mutation at amino acid 153 abolished origin binding, showing it
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Simian virus 40 (SV40) T-antigen is crucial for viral DNA replication and cell transformation.
- Understanding T-antigen's interaction with viral regulatory sequences is key to deciphering its functions.
- Previous studies identified T-antigen variants with impaired DNA replication initiation.
Purpose of the Study:
- To investigate the DNA-binding properties of SV40 T-antigen variants.
- To determine the role of specific T-antigen mutations in DNA binding and viral replication.
- To assess the necessity of origin-binding for T-antigen's transforming function.
Main Methods:
- Analysis of T-antigen binding affinity to SV40 origin of replication sequences in cell lines.
- Site-directed mutagenesis to create SV40 T-antigen mutants with specific amino acid changes.
- Transformation assays using mutated SV40 DNA to assess T-antigen function.
- Immunoprecipitation using a T-antigen-specific monoclonal antibody (PAb 100).
Main Results:
- T-antigens from C2 and C11 cell lines specifically bound to viral origin sequences.
- C6 T-antigen exhibited no specific affinity for viral DNA sequences.
- A single mutation at amino acid position 153 (Asn to Thr) in T-antigen was sufficient to abolish origin-binding.
- The monoclonal antibody PAb 100 recognized wild-type and C6-1 T-antigens but not C6 or C6-2 variants.
- Viral replication function involves properties beyond specific SV40 origin binding.
Conclusions:
- Specific binding to SV40 regulatory sequences is not essential for T-antigen's transforming function.
- Viral replication requires T-antigen properties independent of its origin-binding capability.
- The mutation at amino acid 153 significantly impacts T-antigen's DNA-binding and functional properties.