Biochemical characterization of nuclear and cytoplasmic forms of SV40 tumor antigens encoded by parental and transport-defective mutant SV40-adenovirus 7 hybrid viruses

The biochemical properties of nuclear (nT-ag) and cytoplasmic (cT-ag) SV40 tumor antigens encoded by the defective SV40-adenovirus 7 hybrid virus, PARA, and a mutant [PARA(2cT)j which induces the synthesis of T-ag that is not transported to the nucleus but accumulates in the cytoplasm of infected and transformed cells were compared. Immunoprecipitation and SDS-polyacrylamide gel electrophoresis revealed that parental (nT) PARA and PARA(2cT)-infected and -transformed cells all contained normal-sized large T (92K) and small t(19K) polypeptides. In addition, PARA(2cT)-infected and -transformed cells produced a T-related polypeptide with a slightly higher molecular weight (94K). Cellular fractionation studies demonstrated that both forms of large cT-ag (92K and 94K) were localized predominantly in the cytoplasm of transformed cells; pulse-chase analyses revealed no product-precursor relationship between the two polypeptides. Cell fractionation studies also indicated that cT-ag in PARA(2eT)-transformed cells was not retained in the cytoplasm due to an association with a particulate fraction. PARA-infected cells overproduced T-ag (in comparison to WT SV40) which was stable, resulting in the accumulation of large amounts of T-ag in PARA-infected cells. These results suggest that the transcriptional controls regulating T-ag synthesis by SV40 are not operative with PARA. Quantitation of DNA-binding potentials by radioimmunoassay revealed no differences between nT-ag and cT-ag. Cytoplasmic T-ag from PARA(2cT)-infected cells was phosphorylated to a lesser extent than the nuclear T-ag induced by PARA(nT). Analysis by isoelectric focusing (IEF) in agarose gels of T-ag polypeptides extracted from PARA(nT)- and PARA(2cT)-infected cells revealed a small difference in IEF points (pI 4.4 and pI 4.5, respectively). The possible relationships between phosphorylation, the cT mutation, and the nuclear transport of SV40 T-ag are discussed.