Abstract
An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Qbeta 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 Qbeta 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 Qbeta 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 Qbeta 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 Qbeta RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Qbeta replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Qbeta phage.
Highlights
Recombinations between and within RNA molecules are rare but biologically important events contributing to the evolution and diversity of RNA viruses [1, 2] and generating defective interfering RNAs that attenuate viral infections [3]
RNA Synthesis Is Required for the Generation of Recombinant RNAs by Q Replicase—To investigate requirements of the 3Ј hydroxyl-dependent recombination, we used the experimental approach earlier established for exploring the ability of RNA molecules to self-recombine [7]
Mechanistic Differences between Q Replicase and Poliovirus RNA Polymerase—This article presents results of the first comparative study of intermolecular RNA recombination promoted by RNA-directed RNA polymerases (RdRps) of two different RNA viruses under similar physicochemical conditions, using the same recombination substrates and the same amplification system
Summary
RdRp, RNA-directed RNA polymerase; rNTPs, ribonucleoside triphosphates; PAAE mechanism, primer alignment and extension mechanism; 3Dpol, poliovirus RdRp; RQ RNA, replicable by Q replicase, a non-genomic RNA capable of exponential amplification by Q replicase; 3ЈC fragment, the complementary copy of the 3Ј fragment; nt, nucleotide(s). The spontaneous recombinations between RNA molecules appeared to be several orders of magnitude less frequent than in the presence of Q replicase They did not require free 3Ј hydroxyl groups, indicating that a quite different reaction chemistry was employed, most probably, a Mg2ϩ-catalyzed RNA cleavage generating fragments with 2Ј,3Ј-cyclic phosphate and 5Ј hydroxyl termini, which are cross-ligated. In contrast to the Q replicase-promoted reaction [15] and RdRpindependent poliovirus RNA recombination in vivo [5], only homologous recombinants were produced, even though coding sequences were not involved To explain their observations, the authors proposed a “primer alignment and extension” (PAAE) mechanism, in which no templates are switched. Whereas the PAAE mechanism is efficiently used by 3Dpol to generate recombinant molecules from RNA fragments of opposite polarities, it is totally rejected by Q replicase
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