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Structure and biochemical functions of four simian virus 40 truncated large-T antigens
Abstract:
The structure of four abnormal T antigens which are present in different simian virus 40 (SV40)-transformed mouse cell lines was studied by tryptic peptide mapping, partial proteolysis fingerprinting, immunoprecipitation with monoclonal antibodies, and in vitro translation. The results obtained allowed us to deduce that these proteins, which have apparent molecular weights of 15,000, 22,000, 33,000 and 45,000, are truncated forms of large-T antigen extending to different amounts into the amino acid sequences unique to large-T. The proteins are all phosphorylated, probably at a site between amino acids 106 and 123. The mRNAs coding for the proteins probably contain the normal large-T splice but are shorter than the normal transcripts of the SV40 early region. The truncated large-Ts were tested for the ability to bind to double-stranded DNA-cellulose. This showed that the 33,000- and 45,000-molecular-weight polypeptides contained sequences sufficient for binding under the conditions used, whereas the 15,000- and 22,000-molecular-weight forms did not. Together with published data, this allows the tentative mapping of a region of SV40 large-T between amino acids 109 and 272 that is necessary and may be sufficient for the binding to double-stranded DNA-cellulose in vitro. None of the truncated large-T species formed a stable complex with the host cell protein referred to as nonviral T-antigen or p53, suggesting that the carboxy-terminal sequences of large-T are necessary for complex formation.
Insights
Researchers studied abnormal simian virus 40 (SV40) T antigens, finding they are truncated forms of large-T antigen. Specific truncated forms bind DNA, while others do not, suggesting key DNA-binding regions within large-T.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Simian virus 40 (SV40) large-T antigen plays a crucial role in viral replication and cellular transformation.
- Abnormal forms of T antigens can arise in SV40-transformed cells, potentially altering viral functions.
- Understanding the structure-function relationship of these T antigens is vital for comprehending SV40 pathogenesis.
Purpose of the Study:
- To characterize the structure of four abnormal T antigens found in SV40-transformed mouse cell lines.
- To determine the relationship of these abnormal proteins to the full-length SV40 large-T antigen.
- To investigate the DNA-binding capabilities and p53 interaction of these truncated T antigens.
Main Methods:
- Tryptic peptide mapping and partial proteolysis fingerprinting were used to analyze protein structure.
- Immunoprecipitation with monoclonal antibodies identified specific T antigen forms.
- In vitro translation and DNA-cellulose binding assays assessed protein function.
Main Results:
- Four truncated forms of SV40 large-T antigen (15, 45,000, 22,000, 33,000, and 45,000 Da) were identified.
- These truncated proteins are phosphorylated, likely within amino acids 106-123.
- The 33,000- and 45,000-Da forms bind double-stranded DNA-cellulose, while the smaller forms do not.
- None of the truncated forms stably complexed with p53, indicating C-terminal sequences are required for this interaction.
Conclusions:
- The abnormal T antigens are distinct C-terminal deletion mutants of SV40 large-T.
- A region between amino acids 109 and 272 of SV40 large-T is necessary for double-stranded DNA binding in vitro.
- SV40 large-T antigen's carboxy-terminal sequences are essential for stable complex formation with p53.