Intracellular localization of adenovirus type 5 tumor antigens in productively infected cells

Abstract

The intracellular localization of tumor antigens of human adenovirus type 5 (Ad5) during lytic infection of KB cells has been studied. The cells were pulse labeled with [ 35S]methionine early after infection and early proteins of 58,000 D (58K), 44K,19K,18.5K, and 14K detectable by immunoprecipitation with hamster antitumor serum were assayed for association with cytoplasm, nucleoplasm, chromatin, cytosol, cytoskeleton, and membranes. The 44,000 D (44K) tumor antigen encoded in early region IA (E1A: 0–4.4%) was recovered in approximately equal amounts from cytoplasmic and nucleoplasmic fractions of pulse-labeled cells and within the cytoplasmic compartment was found in the cytosol as well as associated with the cytoskeleton. The E1B-58K (E1B: 4.5–11.2%) antigen was also found to be associated with the cytoplasmic and nucleoplasmic fractions in approximately equal amounts but unlike the ElA-44K showed no affinity for cytoskeletons. Pulse-chase and immunofluorescence experiments suggested the 58K antigen accumulated in the nucleus late in infection. The E1B-19K antigen was found almost exclusively associated with the membrane fraction of infected KB cells and was resolved in polyacrylamide gels into two related species of 18.5K and 19K. Immunofluorescence studies on the ElB 18.5–19K doublet suggested that within a population of infected HeLa cells a small minority seemed to be expressing copious amounts of stainable antigen. Cell fractionation and immunofluorescence studies showed that the E4-14K antigen was a nuclear protein and the only antigen in this study which showed a significant association with a nuclear subfraction composed almost entirely of histones. The implications of these findings for the roles of the Ad5 tumor antigens in lytic infection and transformation are discussed.