Extraction and fingerprint analysis of simian virus 40 large and small T-antigens

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Abstract
A study of SV-40 T[tumor]antigens isolated from productively infected [African green monkey kidney] CV1 cells using a variety of different extraction procedures showed that under some conditions the highest MW form of T-Ag (large-T) isolated comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with large-T from SV-40 transformed [hamster] H65-90B cells. Other faster-migrating forms of large-T are probably generated during the extraction procedure by a protease which is active at low pH, and such forms are probably experimental artifacts. After extraction under conditions which minimize proteolytic degradation of large-T, a further form of T-antigen was isolated; this has an apparent MW weight in the range 15,000-20,000 and is referred to as small-t. Fingerprint analysis of [35S]methionine-labeled SV-40 proteins showed that small-t has 10-12 methionine peptides but large-T has 15-18 methionine peptides. All but 2 of the methionine tryptic peptides present in small-t are also present in large-T. The fingerprint data also showed that T-antigens have no peptides in common with SV-40 VP[viral protein]1. Experiments using reagents which inhibit posttranslational cleavage of encephalomyocarditis virus polyproteins showed that these reagents do not affect the synthesis of small-t and suggest that it is not made by proteolytic cleavage of large-T in vivo. An alternative model, which proposes that large-T and small-t are synthesized independently, is discussed in terms of the fingerprint data and the number of methionine tryptic peptides predicted from the primary sequence of SV-40 DNA.