Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

Abstract

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses.

Author Summary

The rapid evolution of most positive-sense RNA viruses enables them to escape immune surveillance and adapt to new hosts. Genetic variation arises due to their error-prone RNA polymerases and by recombination of viral genomes in co-infected cells. We have developed a novel approach to analyse the poorly understood mechanism of recombination using a poliovirus model system. We characterised the initial viable recombinants and demonstrate the majority are longer than genome length due to an imprecise crossover event that duplicates part of the genome. These viruses are unfit, but rapidly lose the duplicated material and regain full fitness upon serial passage, a process we term resolution. We show this is a replicative recombination process by modifying the fidelity of the viral polymerase, or replication complex coalescence, using methods that have no influence on a previously reported, less efficient, non-replicative recombination mechanism. We conclude that recombination is a biphasic process involving separate generation and resolution events. These new insights into an important evolutionary mechanism have implications for our understanding of virus evolution through partial genome duplication, they suggest ways in which recombination might be modified and provides an approach that may be exploited to analyse recombination in other RNA viruses.