Multiple Barriers to the Evolution of Alternative Gene Orders in a Positive-Strand RNA Virus.

The order in which genes are organized within a genome is generally not conserved between distantly related species. However, within virus orders and families, strong conservation of gene order is observed. The factors that constrain or promote gene-order diversity are largely unknown, although the regulation of gene expression is one important constraint for viruses. Here we investigate why gene order is conserved for a positive-strand RNA virus encoding a single polyprotein in the context of its authentic multicellular host. Initially, we identified the most plausible trajectory by which alternative gene orders could evolve. Subsequently, we studied the accessibility of key steps along this evolutionary trajectory by constructing two virus intermediates: (1) duplication of a gene followed by (2) loss of the ancestral gene. We identified five barriers to the evolution of alternative gene orders. First, the number of viable positions for reordering is limited. Second, the within-host fitness of viruses with gene duplications is low compared to the wild-type virus. Third, after duplication, the ancestral gene copy is always maintained and never the duplicated one. Fourth, viruses with an alternative gene order have even lower fitness than viruses with gene duplications. Fifth, after more than half a year of evolution in isolation, viruses with an alternative gene order are still vastly inferior to the wild-type virus. Our results show that all steps along plausible evolutionary trajectories to alternative gene orders are highly unlikely. Hence, the inaccessibility of these trajectories probably contributes to the conservation of gene order in present-day viruses.