| Summary: | Positive-sense single stranded RNA viruses, like those found within the Enterovirus genus, have error prone polymerases, short replication cycles and high yields that together generate genetic diversity. These advantageous evolutionary mechanisms enable the virus population to survive in changing environments. However, the majority of mutations that are introduced are lethal or deleterious. RNA recombination provides a mechanism that can remove a deleterious mutation and also provides an opportunity to break the link between mutations that are lethal and those that are beneficial in the same genome. The objectives of this investigation were to identify characteristics of the virus that were important in recombination. As genetic exchange in enteroviruses was postulated to be a replicative process, the role of the RNA dependent RNA polymerase (RdRp) was reasoned to be important. This study has benefited from utilising a novel reverse genetics in vitro cell based assay known as CRE-REP. The assay allowed selection and isolation of viable recombinant virus as near to the recombination event as temporally possible. Results indicated that ‘copy choice’ replicative recombination is a biphasic process with distinct template-exchange and resolution events. Manipulating known RNA elements within the viral genome expanded the cell-based assay further. Notably, this investigation suggested that fidelity of the RNA dependent RNA polymerase (RdRp) is a major determinant of recombination frequency. To further validate the cell-based approach, a defined biochemical assay that allowed quantification of the template transfer event was developed in collaboration with Professor Craig Cameron. The results confirmed that the RdRp alone was sufficient for recombination and that fidelity was a major contributing factor to efficiency. The characterised high fidelity RdRp variants (e.g. K359R, G64S) reduced template transfer up to 25-fold. The biochemical assay was extended to investigate the sequence identity required for the template transfer event. These studies form the basis for the further biophysical dissection of a key evolutionary process present in many positive-strand RNA viruses.
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