Just back from the excellent Europic 2016 meeting in Switzerland where Kirsten Bentley “knocked their socks off” with a talk on her recent analysis of the role of sequence identity and RNA structure in genetic recombination in enteroviruses.
Here’s what our friends in the Cameron and Vignuzzi labs tweeted …
Kirsten at Europic 2016
With thanks to Caroline, Urs and Laurent for running a great meeting …
Nucl. Acid. Res.
The fidelity of the virus polymerase influences the rate of genetic recombination between viruses coinfecting the same cell. We used cell-based and new, biochemically-defined, assays to demonstrate that the viral polymerase is necessary and sufficient for the strand-transfer event of RNA virus recombination. Furthermore, the fidelity of the polymerase is critical in determining the efficiency with which recombination occurs; low fidelity polymerases exhibit high recombination rates, and vice versa.
The paper is published in Nucleic Acids Research:
Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination
Andrew Woodman; Jamie J. Arnold; Craig E. Cameron; David J. Evans
Nucleic Acids Research 2016; doi: 10.1093/nar/gkw567
Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3′ non-coding region we have further developed a cell-based assay (the 3′CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process.
Recently accepted for publication
Wood et al., (2014) MosaicSolver: a tool for determining recombinants of viral genomes from pileup data. Nucleic Acids Research (in press)
Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, changes in tropism and possibly transmission across species barriers. Determining whether recombination has occurred and the specific recombination points is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method, using siRNAs and sequence data sampling the viral genome population (cDNA library). Matlab software (MosaicSolver) is available.
Recently accepted for publication
Lowry et al., (2014) Recombination in enteroviruses is a biphasic replicative process involving the generation of greater-than genome length ‘imprecise’ intermediates. PLoS Pathogens DOI: 10.1371/journal.ppat.1004191
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.