Anouk Willemsen

An ancient history of gene duplications, fusions and losses in the evolution of APOBEC3 mutators in mammals

BackgroundThe APOBEC3 (A3) genes play a key role in innate antiviral defense in mammals by introducing directed mutations in the DNA. The human genome encodes for seven A3 genes, with multiple splice alternatives. Different A3 proteins display different substrate specificity, but the very basic question on how discerning self from non-self still remains unresolved. Further, the expression of A3 activity/ies shapes the way both viral and host genomes evolve.ResultsWe present here a detailed temporal analysis of the origin and expansion of the A3 repertoire in mammals. Our data support an evolutionary scenario where the genome of the mammalian ancestor encoded for at least one ancestral A3 gene, and where the genome of the ancestor of placental mammals (and possibly of the ancestor of all mammals) already encoded for an A3Z1-A3Z2-A3Z3 arrangement. Duplication events of the A3 genes have occurred independently in different lineages: humans, cats and horses. In all of them, gene duplication has resulted in changes in enzyme activity and/or substrate specificity, in a paradigmatic example of convergent adaptive evolution at the genomic level. Finally, our results show that evolutionary rates for the three A3Z1, A3Z2 and A3Z3 motifs have significantly decreased in the last 100 Mya. The analysis constitutes a textbook example of the evolution of a gene locus by duplication and sub/neofunctionalization in the context of virus-host arms race.ConclusionsOur results provide a time framework for identifying ancestral and derived genomic arrangements in the APOBEC loci, and to date the expansion of this gene family for different lineages through time, as a response to changes in viral/retroviral/retrotransposon pressure.

Experimental Evolution of Pseudogenization and Gene Loss in a Plant RNA Virus

Viruses have evolved highly streamlined genomes and a variety of mechanisms to compress them, suggesting that genome size is under strong selection. Horizontal gene transfer has, on the other hand, played an important role in virus evolution. However, evolution cannot integrate initially nonfunctional sequences into the viral genome if they are rapidly purged by selection. Here we report on the experimental evolution of pseudogenization in virus genomes using a plant RNA virus expressing a heterologous gene. When long 9-week passages were performed, the added gene was lost in all lineages, whereas viruses with large genomic deletions were fixed in only two out of ten 3-week lineages and none in 1-week lineages. Illumina next-generation sequencing revealed considerable convergent evolution in the 9- and 3-week lineages with genomic deletions. Genome size was correlated to within-host competitive fitness, although there was no correlation with virus accumulation or virulence. Within-host competitive fitness of the 3-week virus lineages without genomic deletions was higher than for the 1-week lineages. Our results show that the strength of selection for a reduced genome size and the rate of pseudogenization depend on demographic conditions. Moreover, for the 3-week passage condition, we observed increases in within-host fitness, whereas selection was not strong enough to quickly remove the nonfunctional heterologous gene. These results suggest a demographically determined “sweet spot” might exist, where heterologous insertions are not immediately lost while evolution can act to integrate them into the viral genome.

EcPV2 DNA in equine genital squamous cell carcinomas and normal genital mucosa

Squamous cell carcinoma (SCC) represents the most common genital malignant tumor in horses. Similar to humans, papillomaviruses (PVs) have been proposed as etiological agents and recently Equine papillomavirus type 2 (EcPV2) has been identified in a subset of genital SCCs. The goals of this study were (1) to determine the prevalence of EcPV2 DNA in tissue samples from equine genital SCCs, penile intraepithelial neoplasia (PIN) and penile papillomas, using EcPV2-specific PCR, (2) to examine the prevalence of latent EcPV2 infection in healthy genital mucosa and (3) to determine genetic variability within EcPV2 and to disentangle phylogenetic relationships of EcPV2 among PVs. EcPV2 DNA was detected in all but one penile SCC (15/16), in all PIN lesions (8/8) and penile papillomas (4/4). Additionally, EcPV2 DNA was demonstrated in one of two metastasized lymph nodes, one contact metastasis in the mouth, two vaginal and one anal lesion. In healthy horses, EcPV2 DNA was detected in 10% (4/39) of penile swabs but in none of vulvovaginal swabs (0/20). This study confirms the presence of EcPV2 DNA in equine genital SCCs and shows its involvement in anal lesions, a lymph node and contact metastases. Latent EcPV2 presence was also shown in normal male genital mucosa. We found that different EcPV2 variants cocirculate among horses and that EcPV2 is related to the Delta+Zeta PVs and is only a very distant relative of high-risk human PVs causing genital cancer. Thus, similar viral tropism and similar malignant outcome of the infection do not imply close evolutionary relationship.

Emergence and Phylodynamics of Citrus tristeza virus in Sicily, Italy

Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.

Genetic Variation and Possible Mechanisms Driving the Evolution of Worldwide Fig mosaic virus Isolates

Fig mosaic virus (FMV) is a multipartite negative-sense RNA virus infecting fig trees worldwide. FMV is transmitted by vegetative propagation and grafting of plant materials, and by the eriophyid mite Aceria ficus. In this work, the genetic variation and evolutionary mechanisms shaping FMV populations were characterized. Nucleotide sequences from four genomic regions (each within the genomic RNAs 1, 2, 3, and 4) from FMV isolates from different countries were determined and analyzed. FMV genetic variation was low, as is seen for many other plant viruses. Phylogenetic analysis showed some geographically distant FMV isolates which clustered together, suggesting long-distance migration. The extent of migration was limited, although varied, between countries, such that FMV populations of different countries were genetically differentiated. Analysis using several recombination algorithms suggests that genomes of some FMV isolates originated by reassortment of genomic RNAs from different genetically similar isolates. Comparison between nonsynonymous and synonymous substitutions showed selection acting on some amino acids; however, most evolved neutrally. This and neutrality tests together with the limited gene flow suggest that genetic drift plays an important role in shaping FMV populations.

Temporal Dynamics of Intrahost Molecular Evolution for a Plant RNA Virus

Populations of plant RNA viruses are highly polymorphic in infected plants, which may allow rapid within-host evolution. To understand tobacco etch potyvirus (TEV) evolution, longitudinal samples from experimentally evolved populations in the natural host tobacco and from the alternative host pepper were phenotypically characterized and genetically analyzed. Temporal and compartmental variabilities of TEV populations were quantified using high throughput Illumina sequencing and population genetic approaches. Of the two viral phenotypic traits measured, virulence increased in the novel host but decreased in the original one, and viral load decreased in both hosts, though to a lesser extent in the novel one. Dynamics of population genetic diversity were also markedly different among hosts. Population heterozygosity increased in the ancestral host, with a dominance of synonymous mutations fixed, whereas it did not change or even decreased in the new host, with an excess of nonsynonymous mutations. All together, these observations suggest that directional selection is the dominant evolutionary force in TEV populations evolving in a novel host whereas either diversifying selection or random genetic drift may play a fundamental role in the natural host. To better understand these evolutionary dynamics, we developed a computer simulation model that incorporates the effects of mutation, selection, and drift. Upon parameterization with empirical data from previous studies, model predictions matched the observed patterns, thus reinforcing our idea that the empirical patterns of mutation accumulation represent adaptive evolution.

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

ABSTRACT Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.

Predicting the stability of homologous gene duplications in a plant RNA virus

Abstract One of the striking features of many eukaryotes is the apparent amount of redundancy in coding and non-coding elements of their genomes. Despite the possible evolutionary advantages, there are fewer examples of redundant sequences in viral genomes, particularly those with RNA genomes. The factors constraining the maintenance of redundant sequences in present-day RNA virus genomes are not well known. Here, we use Tobacco etch virus, a plant RNA virus, to investigate the stability of genetically redundant sequences by generating viruses with potentially beneficial gene duplications. Subsequently, we tested the viability of these viruses and performed experimental evolution. We found that all gene duplication events resulted in a loss of viability or in a significant reduction in viral fitness. Moreover, upon analyzing the genomes of the evolved viruses, we always observed the deletion of the duplicated gene copy and maintenance of the ancestral copy. Interestingly, there were clear differences in the deletion dynamics of the duplicated gene associated with the passage duration and the size and position of the duplicated copy. Based on the experimental data, we developed a mathematical model to characterize the stability of genetically redundant sequences, and showed that fitness effects are not enough to predict genomic stability. A context-dependent recombination rate is also required, with the context being the duplicated gene and its position. Our results therefore demonstrate experimentally the deleterious nature of gene duplications in RNA viruses. Beside previously described constraints on genome size, we identified additional factors that reduce the likelihood of the maintenance of duplicated genes.

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.

2b or not 2b : Experimental evolution of functional exogenous sequences in a plant RNA virus

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.