Assignment of viral proteins to the three double-stranded RNA segments of bacteriophage ϕ6 genome: Translation of ϕ6 messenger RNAs transcribed in vitro

Abstract

Summary Pseudomonas phaseolicola bacteriophage ϕ6 has a double-stranded (ds) RNA genome in three segments (L, M, S) which can serve as templates for in vitro transcription by ϕ6 nucleocapsid. Singlestranded (ss) RNA (l, m, s) synthesized in vitro functioned as messenger RNA of viral proteins in an Escherichia coli cell-free protein-synthesizing system. Each of the three ssRNA species was isolated in virtually pure form and translated, providing a means of determining the polypeptides encoded by each segment. From the analysis of polypeptide products by SDS-polyacrylamide gel electrophoresis, coding assignments of three dsRNA segments were established. The major structural proteins P8 and P9 were shown to be encoded by the S segment transcript (s). The membrane proteins P3, P6, and P10 are encoded by the M segment transcript (m). The nucleocapsid proteins P1, P4, and P7 are probably synthesized by the L-segment transcript (l), but there remained a possibility that P4 and P7 are encoded also by the M-segment transcript (m). The nucleocapsid protein P2 was not synthesized in detectable amounts by transcripts of any segments in our experiments. This protein is known to be synthesized in small amounts in vivo. The lytic enzyme P5 could not be identified owing to the difficulty in separating P5 from products of the endogenous protein-synthesizing activity of E. coli extracts.