Our experimental evolution studies of vaccinia evoke a “gene accordion” model for genome evolution. Localized genomic regions transiently expand to enhance levels of gene expression, while simultaneously providing additional gene copies for sampling mutational space (). Subsequent contractions mitigate the costs of replicating larger genomes while retaining adaptive alleles like H47R that arose during the expansion. Thus, a simple mechanism of recombination-driven genomic expansions and contractions facilitates the rapid evolution of virus populations with otherwise low mutation rates.
Genomic accordion model of poxvirus evolution
Gene accordions do not appear unique to the K3L locus. In addition to the low frequency duplications we detected in vaccinia genomes ( and Table S3
), this model of adaptation through intermediates of transient gene expansion also helps explain a variety of previous observations in the genomes of large double-stranded DNA viruses, including ancient gene family expansions among several poxviruses (McLysaght et al., 2003
) and herpesviruses (Searles et al., 1999
), chemically induced gene amplification in vaccinia (Slabaugh and Mathews, 1986
; Slabaugh et al., 1989
), and a potentially adaptive duplication observed in the myxoma poxvirus (Kerr et al., 2010
). A particularly clear example of such adaptive gene expansions is evident in the genomes of avipoxviruses, which devote as much as 30% of the genome to a small set of large gene families that includes many ankyrin repeat containing genes (Afonso et al., 2000
). Avipoxviruses illustrate an exceptional example of gene duplications facilitating neofunctionalization of individual copies of the amplified gene family, leading to the retention of multiple variants rather than contraction back to a single copy. We propose that sampling a reservoir of low frequency duplications at various genomic regions in the virus population reflects an underappreciated but common mechanism seeding dynamic and adaptive gene expansions in these viruses. Our observations reveal the underlying mechanisms by which such viruses adapt to new, hostile host environments first by rapid gene expansion rather than gene mutation.
These results also reveal that a strength of the experimental evolution strategy is its power to reveal important evolutionary intermediates that could be too fleeting to capture by less frequent sampling or missed all together by studying viruses long after host switches. For example, we could clearly demonstrate that vaccinia genomes underwent a specific K3L gene expansion that exactly correlated with all fitness gains observed during our experiment. However, by 10 passages the K3L gene had already been replaced by the H47R variant in 12% of all K3L genes in one of the replicates. Sampling at a later point could conceivably obscure the accordion-like origins of an H47R-like adaptation as the variant sweeps through the population and replaces large and costlier gene expansions. This duality of adaptation might also be reflected in the different stages of viral transmission between species, including zoonosis. Gene expansions might be favored early in infection when most viral adaptation is centered on overcoming host defenses, as was modeled by our experiment. At later stages of infection, gene expansions are likely to be strongly selected against as viruses compete not only against the initial challenge imposed by host defenses, but also against larger populations of fit viruses carrying the equivalent of an H47R mutation. Future studies will be important to assess not only the generality of this adaptive mechanism, but also to compare the dynamics observed in our experiments to those that occur during natural poxviral infections in future studies.
Despite the rapid gene expansions observed in our study, very few poxviruses sequenced from the wild have been found to harbor recent gene duplications or expansions, with the ankyrin repeat genes in avipoxviruses being a clear exception. While some expansions may be obscured by subsequent contractions, it is also possible that many expansion events have been missed because of an ascertainment bias. Until recently, sequencing methodologies rarely accounted for copy number variation as a source of genetic heterogeneity. Indeed, sequencing of different strains of variola (smallpox) virus with the aim of determining how variola major had a higher fatality rate than variola minor focused primarily on nucleotide substitutions, which revealed less than 2% divergence (Esposito et al., 2006
). While this genetic divergence might be sufficient to explain differential pathogenicity, important instances of copy number variation might have gone undetected in these comparisons. New sequencing technologies greatly increase the prospects of capturing such intermediate states from poxviruses that have undergone minimal passaging in the lab.
Our study also highlights the important role that gene copy number expansion plays in adaptation of genomes with low mutation rates. This mode of adaptation may be especially prevalent when an immediate advantage can be gained via mass action. Examples include selection for resistance to drug therapies against cancer (Drummond et al., 1997
; Schimke, 1986
), for enhanced enzymatic degradation of toxins (Sandegren and Andersson, 2009
), or for overcoming a host antiviral protein like PKR. In these cases, the duplication event is itself non-neutral and can be further acted upon by recombination to drastically alter genome architecture. While rapid evolution of copy number variation is known for some non-virus microbial populations, including Plasmodium
, yeast, and a variety of bacteria (Bergthorsson et al., 2007
; Demuth and Hahn, 2009
; Dunham et al., 2002
; Kugelberg et al., 2010
; Nair et al., 2008
; Pränting and Andersson, 2011
; Stambuk et al., 2009
; Sun et al., 2009
), adaptation via gene expansion in poxviruses represents an unappreciated mechanism of evolution in this large, medically relevant class of DNA viruses (Harrison et al., 2004
). In the cases cited above, copy number expansions may also be poised for adaptive contractions, but that unfold on longer time scales, thus obscuring a ‘genomic-accordion’ mechanism revealed here by vaccinia virus (but also see Pränting and Andersson, 2011
Some of the passage 10 viruses we assayed have a 7-10% expansion in genome size purely by virtue of a single gene expansion. In a 200 kb genome, this is a noteworthy adaptation for its major alteration of genomic structure. Our findings also indicate that a diversity of adaptive strategies - copy number variation in viruses with low mutation rates and large genomes versus nucleotide substitution - falls well within the viral kingdom. It seems likely that this mode of adaptation is applicable to many other viruses that have similarly low mutation rates and relatively limited restrictions on genome size. Exceptions are likely to be viruses that have low mutation rates but strict limits on genome size due to a highly constrained capsid structure (e.g., adenoviruses and certain bacteriophages). It will be informative to determine whether other large DNA viruses like herpesviruses, which replicate in the nucleus and also show evidence of gene expansions (Searles et al., 1999
), follow similar gene-accordion dynamics as the poxviruses, which replicate in the cytoplasm of host cells.
There are striking parallels between the challenges imposed on viruses by exposure to new batteries of innate defense genes and the challenges imposed on organisms adapting to strong selective pressure, like bacteria grown under stress-inducing conditions (Andersson et al., 1998
; Cairns et al., 1988
; Foster, 1994
; Hastings et al., 2000
; Hendrickson et al., 2002
). In each of these cases, a significant step in the adaptive process involves rapid and short-lived gene expansions that permit the selection of ‘escape’ variants. Our study thus reveals a previously unappreciated insight into the dynamics of Red Queen conflicts between pathogens and hosts (Elde and Malik, 2009
; Emerman and Malik, 2010
; Meyerson and Sawyer, 2011
). Traditionally viewed primarily as substrates for rapid selection of non-synonymous codon substitutions, these interactions may also depend on adaptive gene amplifications that are overlooked due to their transience or difficulty of detection (). Indeed, ‘gene-accordions’ may be a critical feature of many genetic conflicts involving strong selection.