Translation of poliovirus rna in vitro: Changes in cleavage pattern and initiation sites by ribosomal salt wash

Abstract

Translation of poliovirus RNA in two different micrococcal nuclease-treated lysates (from rabbit reticulocytes and HeLa cells) yields a distinctive profile of poliovirus proteins. Synthesis is stimulated in both cases by the addition of ribosomal salt wash from either reticulocytes or from polio-infected or uninfected HeLa cells. When reticulocyte lysates are stimulated with ribosomal salt wash from HeLa cells, however, changes in the electrophoretic pattern of newly synthesized products occur so as to decrease the number of polypeptides produced and to increase the yield of products which correspond to those seen in poliovirus-infected cells. These changes appear to be the result of cell-specific proteases present in ribosomal salt wash which affect the cleavage pattern of the polio proteins. Additional ribosomal salt wash effects on translation products have been distinguished by analysis of N-formyl[ 35S]methionine-labeled tryptic peptides produced by initiation of translation. HeLa cell extracts utilize two initiation sites for translation of poliovirus RNA at efficiencies which are dependent upon Mg 2+ concentration. Reticulocyte lysates utilize predominantly one initiation site at all Mg 2+ concentrations in reaction mixtures not supplemented with exogenous ribosomal salt wash. However, polio-infected cell ribosomal salt wash preferentially stimulates translation at the second site in the reticulocyte system.