Abstract
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.
Highlights
Recombination in single-stranded, positive-sense RNA viruses is a relatively poorly understood driver of extensive genetic change. This group of viruses, typified by poliovirus, exist as viral quasispecies as a consequence of misincorporations by their error-prone RNA dependent RNA polymerases (RdRp) during genome replication coupled with strand transfer events that create hybrids between two viruses replicating in the same cell
Studies of paralysis induced by vaccine derived recombinant polioviruses (VDRP)––in which the live attenuated Sabin vaccine strain has recombined with a co-circulating species C enterovirus––have demonstrated the importance of recombination in vivo [7]
We have demonstrated that this mutation decreased recombinant yield in an intertypic cis-acting replication element (CRE)-REP assay by ∼20-fold whereas the addition of ribavirin, which enhances the polymerase error rate, increased recombinant yield in the same assay [11]
Summary
Recombination in single-stranded, positive-sense RNA viruses is a relatively poorly understood driver of extensive genetic change. The genome can essentially be considered modular, consisting of structural (P1) and replication (P2 and P3) components, together with the flanking translation and replication determinants occupying the 5 and 3 NCRs [3,4] This modularity is emphasized in in vitro studies in which viable recombinants have been engineered or selected [5,6]. Recombinant forms––defined by serotype according to their capsid proteins––have been shown to emerge, prevail and disappear in temporal epidemiological surveys of globally-distributed serotypes [8,9,10] In these examples the recombinants are pathogenic (generally associated with poliomyelitis, a range of acute flaccid paralyses or viral encephalitis) and demonstrate maintenance and transmission of the capsid in the population by a range of non-structural ‘modules’ from genetically-related but divergent viruses. An improved mechanistic understanding of recombination is required, both to comprehend the process as a driver of evolutionary change and to develop strate-
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