A complete plasmid-based complementation system for RNA coliphage Qβ: Three proteins of bacteriophages Qβ (Group III) and SP (Group IV) can be interchanged

Our laboratory has established a bacteriophage Qβ cDNA-containing plasmid system in which virtually all coding defects present within the 4217 nucleotide Qβ genome can be complemented in trans. In this system, Qβ minus strand RNAs are constitutively transcribed from plasmid cDNA by Escherichia coli RNA polymerase. Replication of these minus strands results in the synthesis of Qβ plus RNA, thereby triggering an infectious cycle in which Qβ phage particles are generated. Genetically engineered Qβ genome mutations that result in defective viral proteins can be complemented in trans by the products of one or more Qβ helper plasmids that express either: (1) Qβ maturation protein, which can complement defects in the Qβ maturation cistron (nucleotides 61 to 1320); (2) Qβ readthrough protein, which can complement defects in the readthrough cistron (nucleotides 1344 to 2330); or (3) Qβ replicase, which can complement defects in the replicase cistron (nucleotides 2352 to 4118). Each plasmid component of this system contains a unique origin of replication and carries a different antibiotic gene, thereby enabling all combinations of these plasmids to coexist in the same host. We have further developed a second series of helper plasmids that generate the corresponding viral proteins of the related group IV RNA phage SP. Each of these SP helper proteins can complement respective defects within the Qβ genome with efficiencies similar to those observed for the Qβ helper proteins. It is now possible to supply functional Qβ or SP proteins in trans to examine Qβ genomes that contain protein coding defects for their ability to synthesize Qβ proteins, replicate Qβ RNA, assemble virions, and/or lyse the host cell.