Genetic recombination in positive-strand RNA viruses is a significant evolutionary mechanism that drives the creation of viral diversity by the formation of novel chimaeric genomes. The process and its consequences, for example the generation of viruses with novel phenotypes, has historically been studied by analysis of the end products. More recently, with an appreciation that there are both replicative and non-replicative mechanisms at work, and with new approaches and techniques to analyse intermediate products, the viral and cellular factors that influence the process are becoming understood. The major influence on replicative recombination is the fidelity of viral polymerase, although RNA structures and sequences may also have an impact. In replicative recombination the viral polymerase is necessary and sufficient, although roles for other viral or cellular proteins may exist. In contrast, non-replicative recombination appears to be mediated solely by cellular components. Despite these insights, the relative importance of replicative and non-replicative mechanisms is not clear. Using single-stranded positive-sense RNA viruses as exemplars, we review the current state of understanding of the processes and consequences of recombination.

Coronavirus subgenomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving transcription regulatory sequences (TRSs) located in the 5= leader sequence (TRS-L) and upstream of each structural and group-specific gene (TRS-B). Several gammacoronaviruses including infectious bronchitis virus (IBV) contain a putative open reading frame (ORF), localized between the M gene and gene 5, which is controversial due to the perceived absence of a TRS. We have studied the transcription of a novel sgmRNA associated with this potential ORF and found it to be transcribed via a previously unidentified noncanonical TRS-B. Using an IBV reverse genetics system, we demonstrated that the template-switching event during intergenic region (IR) sgmRNA synthesis occurs at the 5= end of the noncanonical TRS-B and recombines between nucleotides 5 and 6 of the 8-nucleotide consensus TRS-L. Introduction of a complete TRS-B showed that higher transcription levels are achieved by increasing the number of nucleotide matches between TRS-L and TRS-B. Translation of a protein from the sgmRNA was demonstrated using enhanced green fluorescent protein, suggesting the translation of a fifth, novel, group-specific protein for IBV. This study has resolved an issue concerning the number of ORFs expressed by members of the Gammacoronavirus genus and proposes the existence of a fifth IBV accessory protein. We confirmed previous reports that coronaviruses can produce sgmRNAs from noncanonical TRS-Bs, which may expand their repertoire of proteins. We also demonstrated that noncanonical TRS-Bs may provide a mechanism by which coronaviruses can control protein expression levels by reducing sgmRNA synthesis.

A phylogenetically conserved RNA structure within the NS5B coding region of hepatitis C virus functions as a cis-replicating element (CRE). Integrity of this CRE, designated SL9266 (alternatively 5BSL3.2), is critical for genome replication. SL9266 forms the core of an extended pseudoknot, designated SL9266/PK, involving long distance RNA–RNA interactions between unpaired loops of SL9266 and distal regions of the genome. Previous studies demonstrated that SL9266/PK is dynamic, with ‘open’ and ‘closed’ conformations predicted to have distinct functions during virus replication. Using a combination of site-directed mutagenesis and locked nucleic acids (LNA) complementary to defined domains of SL9266 and its interacting regions, we have explored the influence of this structure on genome translation and replication. We demonstrate that LNAs which block formation of the closed conformation inhibit genome translation. Inhibition was at least partly independent of the initiation mechanism, whether driven by homologous or heterologous internal ribosome entry sites or from a capped message. Provision of SL9266/PK in trans relieved translational inhibition, and mutational analysis implied a mechanism in which the closed conformation recruits a cellular factor that would otherwise suppresses translation. We propose that SL9266/PK functions as a temporal switch, modulating the mutually incompatible processes of translation and replication.

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