Viral RNA-directed RNA Polymerases Use Diverse Mechanisms to Promote Recombination between RNA Molecules

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qβ phage replicase and the poliovirus 3Dpol protein, to promote RNA recombination through a primer extension mechanism. The substrates of recombination were fragments of complementary strands of a Qβ phage-derived RNA, such that if aligned at complementary 3′-termini and extended using one another as a template, they would produce replicable molecules detectable as RNA colonies grown in a Qβ replicase-containing agarose. The results show that while 3Dpol efficiently extends the aligned fragments to produce the expected homologous recombinant sequences, only nonhomologous recombinants are generated by Qβ replicase at a much lower yield and through a mechanism not involving the extension of RNA primers. It follows that the mechanisms of RNA recombination by poliovirus and Qβ RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qβ replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qβ phage.