Minus-strand RNA Synthesis by the Segmented Double-stranded RNA Bacteriophage Phi 6 Requires Continuous Protein Synthesis

Bacteriophage phi 6 contains three dsRNA chromosomes. Strand-separating agarose gels were used to study plus- and minus-strand synthesis in vivo and the effect of protein synthesis inhibitors. Analysis of phi 6 RNA synthesis shows low levels of all three dsRNAs and ssRNAs at 10 min, increasing label uptake into all RNAs except the large message from 20 to 60 min, and a greater abundance of medium and small messages than large mRNAs at late times. Isoconformers of the small message are synthesized throughout infection. Northern analysis suggests that large messages made early may persist to direct continuing translation of L-segment-encoded transcription and replication proteins. The time course of phi 6 minus-strand RNA synthesis in vivo, in the absence of background label in host RNAs, is reported for the first time. Label in minus strands is detected only after heat denaturation of RNA samples and appears sequentially in the small, medium, and large strands beginning at 20 min. At both early and late times, chloramphenicol arrests minus-strand synthesis rapidly and all three mRNAs accumulate. The results are consistent with the reovirus asynchronous model for dsRNA viral replication: plus ssRNAs made first are used as templates for minus-strand synthesis. They also indicate that replication protein(s) acts stoichiometrically.