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

Abstract

An earlier developed purified cell-free system was used to explore the potential of two RNA-directed RNA polymerases (RdRps), Q␤ 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 Q␤ 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 Q␤ 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 Q␤ 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 Q␤ RdRps are quite different. The data favor an RNA transesterification reaction catalyzed by a conformation acquired by Q␤ replicase during RNA synthesis and provide a likely explanation for the very low frequency of homologous recombination in Q␤ phage.Recombinations (sequence exchange and rearrangements) 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). In contrast to splicing and other types of regular RNA rearrangements, recombinations occur without apparent sequence or structure specificity (1, 2). There are indications that recombination may occur between cellular RNAs (4 -6), eventually resulting, by means of reverse transcription and integration, in alterations in the chromosomal DNA. Spontaneous Mg 2ϩ -catalyzed rearrangements in RNA sequences (7) might have been a mechanism for evolution in the prebiotic RNA world and might have evolved into contemporary sequencespecific ribozyme-catalyzed reactions (8, 9).RNA recombination was discovered more than 40 years ago as an exchange of genetic markers between polioviruses (10, 11), and since then similar approaches were used to demonstrate that genomes of RNA viruses of animals, plants, and bacteria are all capable of recombination (2, 4, 12, 13). However, such in vivo experiments utilizing living cells, as well as in vitro studies that used crude cell lysates could not uncover the underlying molecular mechanisms or even definitely answer the question if recombining entities were RNA molecules themselves or their cDNA copies, because every living cell contained enzymes capable of reverse transcription and appropriate dNTP substrates. It became evident that further progress in this field depended on the availability of adequate in vitro systems whose composition and other parameters can be strictly controlled by the experimenter (2, 14).The first example of such a sort has been the cell-free system employing purified Q␤ replicase, RNA-directed RNA polymerase (RdRp) 1 of bacteriophage Q␤ (15). The system...

Genomics of Bacterial and Archaeal Viruses: Dynamics within the Prokaryotic Virosphere

“…Such studies have indicated that (i) frequent base substitutions lead to adaptations to changing environmental conditions (20), (ii) deletions as well as duplications occurring by random recombination are readily introduced into the ssRNA of leviviruses in response to various deliberate changes to the genome sequence and structure (161,192), and (iii) genomic defects can be repaired by illegitimate as well as less frequent homologous recombination occurring as a result of transesterification and template switching, respectively (47,196).…”

Ribozyme Activity of RNA Nonenzymatically Polymerized from 3′,5′-Cyclic GMP

“…RNA provides, for the time being, the only plausible solution to self-generation, as allowed by self-polymerization and by a variety of self-catalytic processes. Pioneering reports of RNA recombination have appeared [7][8][9][10][11][12]. With the possible exception of the systems described in [13][14][15], the reactions reported so far are not simple and robust enough to be of likely prebiotic relevance.…”

Mechanisms of viral emergence

“…At low and high fitness values significant fluctuations of fitness values have been observed. This figure is based on previously published data and concepts [4, 9, 11–14, 16, 17, 19, 21, 25, 29, 31, 35, 41, 57, 59–61, 66, 71, 74]. …”