Synthesis and processing of sindbis virus nonstructural proteins in vitro

Polyadenylated 42 S genome RNA of Sindbis virus was extracted from purified virions, selected by binding to oligo(dT)-cellulose, and used to direct cell-free protein synthesis in nuclease-treated reticulocyte lysates. The translation products comprised a series of polypeptides which included distinct species with apparent molecular weights of 250, 205, 155,145, 89, 76, and 60 × 10 3. Their interrelationships were examined by analysis of their kinetics of synthesis, peptide maps, and incorporation of radioisotopic label from formyl[ 35S]methionyl-tRNA f. Polypeptides p89, p76, and p60 were unique, nonoverlapping, and stable. However, their sequences were contained in one or more of the larger products p250, p205, p145, which comprised a set of overlapping polypeptides that shared a common N-terminus. Radioisotope chase experiments showed that p250, p205, p155, and p145 were unstable; that p89, p76, and p60 were generated by proteolytic processing of larger precursors; and that the RNA regions encoding these three stable products were translated in the order: p60, p89, p76. On the basis of their electrophoretic mobilities and peptide maps, p89 and p76 were indistinguishable from two polypeptides found in Sindbis virus-infected cells and are candidates to be components of the Sindbis virus RNA-dependent RNA polymerases. When translation was performed under conditions that were designed to inhibit proteolysis, the accumulation of p89 and p76 was prevented, and that of p60 was severely inhibited. Under certain translation conditions, heterodisperse products larger than p250 accumulated. Even after thermal denaturation of RNA, only small amounts of the viral structural proteins were synthesized, indicating that the 3′-terminal third of the 42 S RNA genome was essentially unavailable for independent translation.