Differentiation between minus- and plus-strand synthesis: polymerase activity of dsRNA bacteriophage phi 6 in an in vitro packaging and replication system.
Empty procapsids of the segmented dsRNA virus phi 6, produced in Escherichia coli from a cloned L genome segment, package plus-strand phi 6 ssRNA genomic segments, synthesize minus strands, and transcribe the newly formed dsRNA templates. Procapsids can be restricted to minus-strand synthesis by high concentrations of CaCl2 or low concentrations of nucleotides, enabling us to separate the viral minus-strand (replication) and plus-strand (transcription) RNA-dependent RNA polymerase activities in vitro. Reaction conditions for minus-strand synthesis were optimized. Plus-strand synthesis by procapsids could be activated by binding of purine nucleoside triphosphates to a low-affinity NTP-binding site. The second 5'-terminal nucleotide of the phi 6 plus-sense ssRNA L genomic segment is important for determining the level of transcription of that segment and the generation of infectious procapsids.
van Dijk
A A
AA
Department of Biosciences, University of Helsinki, Finland.
Frilander
M
M
Bamford
D H
DH
D017126:Bacteriophage phi 6 / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli; D016679:Genome, Viral; D011685:Purine Nucleotides / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D014158:Transcription, Genetic; D014544:Uridine Triphosphate / Q000378:metabolism; D014779:Virus Replication
D011685:Purine Nucleotides; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D012321:DNA-Directed RNA Polymerases; D014544:Uridine Triphosphate