Effects of Different RNAs and Components of the Cell-Free System on In Vitro Synthesis of Sindbis Viral Proteins

Effects of Different RNAs and Components of the Cell-Free System on In Vitro Synthesis of Sindbis Viral Proteins Ranieri Cancedda 1 , Richard Swanson and Milton J. Schlesinger a Department of Microbiology, Division of Biology and Biomedical Sciences, Washington University, St. Louis, Missouri 63310 ABSTRACT Cell-free extracts from Krebs ascites cells and rabbit reticulocytes synthesized a variety of viral-specific proteins when programmed with several different kinds of Sindbis viral RNAs. The RNAs included purified virion RNA (42 S ) and two species (26 S and “33 S ”) of purified intracellular viral messenger RNAs from viral-infected BHK cells. Proteins formed in vitro were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rate-zonal centrifugation in urea-sucrose gradients, two-dimensional tryptic peptide fingerprints, and immunoprecipitation with rabbit anti-Sindbis virus serum. The only major identifiable protein formed in vitro was viral capsid, but the relative amount of capsid produced was determined by the mRNA, the source of cell-free extract, and the components of the cell-free system. Virion RNA directed synthesis of larger-molecular-weight proteins than did intracellular viral RNAs, and some of this protein was distinct from that formed by the smaller viral RNAs. Indirect evidence is presented for in vitro synthesis of viral envelope proteins.