Modulation of the expression of poliovirus proteins in reticulocyte lysates

Abstract

The translation of poliovirus RNA into specific viral proteins in mRNA-dependent reticulocyte lysates (MDLs) was found to be highly dependent on individual lysate preparations. Under optimal conditions. the first polypeptide detected was always P3-1b (formerly NCVP 1b). the product of the 3′ portion of the poliovirus genome; the formation of P1-1a (formerly NCVP 1a) followed as shown by time-course and pulse-chase experiments. However, some lysates synthesized little or no P1-1a despite their ability to synthesize P3-1b and to translate normally other cellular and viral mRNAs. When an MDL competent in synthesizing P1-1a was diluted ca. twofold, while maintaining optimal concentrations of salts, tRNA, DTT, creatine phosphate, and amino acids, P1-1a formation was virtually eliminated, while the synthesis of P3-1b, presumably as a consequence of a more downstream initiation, was maintained. The synthesis of P1-1a in a diluted MDL was restored, and P3-1b synthesis suppressed, by the addition of a S 10 fraction prepared from uninfected or virus-infected HeLa cells. Nuclease treatment and dialysis of the S 10 fraction did not inhibit its activity. These findings indicate that individual MDLs either possess limiting quantities of, or occasionally are deficient in, a factor(s) that promotes the utilization of the presumed 5′ proximal initiation site (the AUG at nucleotide position 781–783) and that a homologous factor(s) exists in HeLa cells. The implication of these findings for the strategy of poliovirus replication is discussed.