Purification and characterization of Simian Virus 40 large T antigen

Abstract

Simian Virus 40 (SV40) is a small DNA virus that multiplies lytically in the rhesus monkey, and in cultured monkey cells. SV40 also causes tumors when injected into newborn hamsters, and transforms cultured hamster, rat, mouse, and human cells into tumor-like cells. Large T antigen is a viral-coded protein that is synthesized early in the lytic cycle, and is expressed continuously in SV40-induced tumors and SV40-transformed cells. In the lytic cycle, T antigen stimulates cellular RNA and DNA synthesis, regulates the transcription of its own gene, and initiates SV40 replication. T antigen is also necessary for the induction and maintenance of cell transformation. To better understand the multiple properties of T antigen, it was purified and several of its chemical and physical properties were characterized. Preliminary investigations were done with T antigen from the mKSA cell line, a line of SV40-transformed mouse cells. Characterization of mKSA nuclear extracts by sucrose step-gradient centrifugation and gel filtration chromatography showed that T antigen existed as both large and small molecular-weight species. The nuclear extracts also contained significant amounts of nucleic acid, predominantly RNA, which adversely affected some T antigen purification procedures.T antigen from SV80 cells, a line of SV40-transformed human fibroblasts, was purified to near homogeneity by a procedure that involved nuclear isolation and extraction, ultracentrifugation, and blue-agarose, Phenyl-Sepharose, heparin-agarose, and DEAE Bio-Gel chromatographic steps. T antigen from freshly prepared nuclear extracts sedimented as a tetramer during glycerol gradient centrifugation. Storage of the nuclear extract at -20(DEGREES)C converted the T antigen to a monomeric species. Immunoprecipitation of T antigen followed by SDS-electrophoresis showed that SV80 cellular extracts contained 91-, 89-, 87-, 83-, 81-, and 78-kDa forms of T antigen. The 91-kDa T antigen was detected only when the cellular extraction buffer contained TPCK or TLCK. It is proposed that the 91-kDa T antigen is produced by the covalent modification of the 89-kDa T antigen by TPCK or TLCK. Also, previously unrecognized conditions that produce the proteolytically cleaved 83-, 81-, and 78-kDa T antigens were discovered.