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Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants
Abstract:
We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.
Insights
Simian virus 40 (SV40) T-antigen studies reveal distinct regions for DNA origin binding and ATPase activity. ATPase activity correlates with viral replication and cell transformation, but origin binding alone does not suffice.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Simian virus 40 (SV40) T-antigen plays crucial roles in viral replication and cellular transformation.
- Understanding the functional domains of T-antigen is key to elucidating its mechanisms of action.
Purpose of the Study:
- To map the specific regions of SV40 T-antigen responsible for DNA origin binding and ATPase activity.
- To determine the correlation between these activities and viral replication, cellular transformation, and host gene activation.
Main Methods:
- Analysis of T-antigens from SV40 A gene deletion mutants.
- Characterization of mutant T-antigen structures using immunoprecipitation.
- Assay of ATPase activity and SV40 origin DNA binding using immobilized T-antigens.
Main Results:
- SV40 T-antigen sequences for origin binding map between 0.54 and 0.35 map units.
- Sequences between 0.24 and 0.20 map units are required for ATPase activity.
- ATPase activity correlated with viral replication and transformation, while origin binding alone did not.
Conclusions:
- Distinct functional domains exist within the SV40 T-antigen for DNA binding and ATPase activity.
- ATPase activity is essential for SV40 replication and transformation, but origin binding is insufficient on its own.
- Neither activity directly correlated with the activation of rRNA genes or host DNA synthesis.