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.

Keywords: experimental evolution; gene duplication; genome stability; virus evolution.

Fig. 1.—

Schematic representation of the different TEV genotypes containing gene duplications. The wild-type TEV (A) codes for 11 mature peptides, including P3N-PIPO embedded within the P3 protein at a +2 frameshift. Five different viral genotypes containing a single gene duplication were constructed. Second copies of HC-Pro (B), NIa-Pro (C), and NIb (D) were introduced between P1 and HC-Pro. A second copy of NIb was also introduced before P1 (D). And a second copy of CP was introduced between NIb and CP (E). For simplification P3N-PIPO is only drawn at the wild-type TEV.

Fig. 2.—

Deletion detection along the evolution experiments. RT-PCR was performed on the region containing a duplication in the viral genotypes (A–D). Either an intact duplicated copy (white boxes), a deletion together with an intact duplicated copy (light-grey boxes), or a partial or complete loss of the duplicated copy (dark-grey boxes) were detected.

Fig. 3.—

The reduction in genome size over time. The different panels display how the genome size of the different viral genotypes with gene duplications (A–D) changes along the evolution experiments. The dotted grey lines indicate the genome sizes of the wild-type virus (below) and the ancestral viruses (above). The genome sizes of the 3-week lineages are drawn with dashed black lines and open symbols, and those of the 9-week lineages are drawn with continuous blue lines and filled symbols.

Fig. 4.—

Within-host competitive fitness of the evolved and ancestral lineages. Fitness (W), as determined by competition experiments and RT-qPCR of the different viral genotypes with respect to a common competitor; TEV-eGFP. The ancestral lineages are indicated by filled circles and the evolved lineages by open circles. The different viral genotypes are color coded, where the wild-type virus is drawn in green. The asterisks indicate statistical significant differences of the evolved lineages as compared with their corresponding ancestral lineages (t-test with Holm–Bonferroni correction).

Fig. 5.—

Virus accumulation of the evolved and ancestral lineages. Virus accumulation, as determined by accumulation experiments and RT-qPCR at 7 dpi of the different viral genotypes. The ancestral lineages are indicated by filled circles and the evolved lineages by open circles. The different viral genotypes are color coded, where the wild-type virus is drawn in green. The asterisks indicate statistical significant differences of the evolved lineages as compared with their corresponding ancestral lineages (t-test with Holm–Bonferroni correction).

Fig. 6.—

The relationship between genome size and within-host competitive fitness. The pink filled circle represents the within-host competitive fitness of the ancestral TEV-NIaPro₂-NIaPro₈ and the green filled circle that of the ancestral wild-type TEV. The black open circles represent the evolved 3-week (right) and 9-week (left) TEV-NIaPro₂-NIaPro₈ lineages. The evolved 9-week lineages, that contain genomic deletions, have a significant higher within-host competitive fitness (Mann–Whitney U = 4.5, P < 0.001) than the evolved 3-week lineages without deletions. A linear regression has been drawn to emphasize the trend in the data.

Fig. 7.—

Genomes of the ancestral and evolved lineages. Mutations were detected using NGS data of the evolved virus lineages as compared with their ancestral lineages. The square symbols represent mutations that are fixed (>50%) and the circle symbols represent mutations that are not fixed (<50%). Filled symbols represent non-synonymous substitutions and open symbols represent synonymous substitutions. Black substitutions occur only in one lineage, whereas color-coded substitutions are repeated in two or more lineages, or in a lineage from another virus genotype. Note that the mutations are present at different frequencies as reported by SAMtools. Grey boxes indicate genomic deletions in the majority variant.