Biosynthesis, Immunological Specificity, and Intracellular Distribution of the Simian Virus 40-Specific Protein Induced by the Nondefective Hybrid Ad2 + ND 1

Abstract

Ad2+ND1, a nondefective hybrid virus containing a segment of the early resion of SV-40 DNA covalently inserted into the human adenovirus 2 genome, enhances the growth of human adenoviruses in simian cells and induces the SV-40 U antigen. This hybrid previously was shown to code for a 28,000 (28K) MW protein not present in wild-type adenovirus 2-infected cells. By radioimmunoprecipitation using sera from hamsters bearing SV-40 specific tumors, the AD2+ND1-induced 28K protein is shown to be SV-40 specific. This Ad2+ND1-induced protein in synthesized as a 30K MW precursor, which is detectable only when infected cells are pulse-labeled in the presence of the protease inhibitor tosylamino phenylethyl chloromethyl ketone. Upon fractionation of labeled cell extracts, about 80% of the 28K protein is found in the plasma membrane fraction, whereas the remaining 20% is associated with the outer nuclear membrane. This protein is not detectable either in the nucleus or in the cytoplasm. Blockage of proteolytic cleavage by tosylamino phenylethyl chloromethyl ketone did not alter the topographic distribution of this SV40 specific protein, although the amount of the precursor protein in the outer nuclear membrane ineased 4-fold while that in the plasma membrane was proportionately decreased. The 28K protein is probably transferred from the outer nuclear membrane to the plasma membrane after posttranslational cleavage of the 30K precursor polypeptide. These data offer further support to the proposal that the 28K protein contains the determinants for SV-40 U antigen and is responsible for SV-40 enhancement of adenovirus growth in simian cells.