Recombination in enteroviruses: the genetics, cell biology and biochemistry of a biphasic replicative mechanism of virus evolution

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Many of the most important human and animal virus pathogens have positive strand RNA genomes. These include, for example, poliovirus, foot and mouth disease virus and deformed wing virus of honeybees. The majority of these RNA viruses evolve very rapidly, generating large populations of highly divergent progeny. This variation helps the virus evade the host immune system - including escaping immunity induced by vaccines - and may enable the virus to spread more efficiently to new hosts, including cross-species transfer. The variation in the virus population is due to two things; the virus has an error-prone polymerase enzyme which, through mis-incorporation, results in imprecise copying during virus replication. This mis-incorporation can result in up to 0.1% divergence in newly synthesized virus genomes. Secondly, if two related viruses co-infect the same cell, the virus can recombine. Recombination facilitates very much more extensive changes of the virus genome - up to 60% in many cases. Recombinant viruses are essentially hybrids, with part of the virus genome derived from one parent, and part from the other parent. Clearly, by combining such extensive regions of two different viruses there are opportunities for very significant changes in the phenotype i.e. the host range, tissues tropism or pathogenic potential, of the resulting virus.Our laboratory has demonstrated the evolution of a virulent recombinant form of deformed wing virus of honeybees which appears to be associated with global disease. Other studies have shown the evolution of neurovirulent poliovirus in a poorly vaccinated population following recombination with a related co-circulating virus. The error-prone polymerases of positive strand RNA viruses are well characterised. In contrast, the process of recombination is only poorly understood. We have developed an assay that, for the first time, allows the recombination process to be divided into two parts - an initial strand transfer event and a secondary resolution event that markedly increases the fitness of the recombinant virus. We propose to use this assay to provide the first detailed analysis of the mechanism of recombination.Our assay unequivocally demonstrates that the generation of a recombinant virus goes via an intermediate in which there are duplications of parts of the virus genome. "Evolution by duplication" is one of the fundamental processes in virus evolution, in which individual genes are duplicated and then can subsequently independently evolve through acquisition of point mutations. The assay we have developed allows this process to be studied experimentally. We will use enteroviruses as a model system; these viruses are generally well-characterised, there are good laboratory systems for their analysis and they are representative of the types of viruses of humans and animals in which recombination is both observed and problematic. We have over 25 years experience studying this group of viruses, in humans and animals.We will study how the sequence of the virus contributes to the frequency and site of recombination. We will analyse where this process occurs in cells and the contribution made to this event by defined cellular proteins. Finally we will investigate the biochemical mechanism involved in the primary recombination (strand transfer) and resolution events. These studies will contribute to an understanding of the fundamental mechanism of the recombination process which is a major driving force in virus evolution. Additionally, by defining the viral and cellular proteins that are involved it will enable the future control, exploitation or inhibition, of recombination - for example, in vaccines of the future.