Willemsen, A.,Zwart, M. P.,Ambros, S.,Carrasco, J. L.,Elena, S. F.

Horizontal gene transfer (HGT) is pervasive in viruses and thought to be a key mechanism in their evolution. On the other hand, strong selective constraints against increasing genome size are an impediment for HGT, rapidly purging horizontally transferred sequences and thereby potentially hindering evolutionary innovation. Here, we explore experimentally the evolutionary fate of viruses with simulated HGT events, using the plant RNA virus Tobacco etch virus (TEV), by separately introducing two functional, exogenous sequences to its genome. One of the events simulates the acquisition of a new function though HGT of a conserved AlkB domain, responsible for the repair of alkylation or methylation damage in many organisms. The other event simulates the acquisition of a sequence that duplicates an existing function, through HGT of the 2b RNA silencing suppressor from Cucumber mosaic virus. We then evolved these two viruses, tracked the maintenance of the horizontally transferred sequences over time, and for the final virus populations, sequenced their genome and measured viral fitness. We found that the AlkB domain was rapidly purged from the TEV genome, restoring fitness to wild-type levels. Conversely, the 2b gene was stably maintained and did not have a major impact on viral fitness. Moreover, we found that 2b is functional in TEV, as it provides a replicative advantage when the RNA silencing suppression domain of HC-Pro is mutated. These observations suggest a potentially interesting role for HGT of short functional sequences in ameliorating evolutionary constraints on viruses, through the duplication of functions.