La emergencia viral como consecuencia de la interacción entre las variabilidades genéticas del virus y huésped

La variabilidad genética en las poblaciones de huéspedes para la susceptibilidad al patógeno es esencial para la propagación de un virus emergente. Los modelos matemáticos predicen que la tasa de difusión del patógeno se frena a medida que aumenta la frecuencia y la diversidad de alelos de resistencia en la población huésped148,230. Sin embargo, las pruebas experimentales de esta hipótesis son escasas. Por ese motivo, este trabajo aborda la cuestión de cuánta heterogeneidad genética del huésped es necesaria para el cambio del destino evolutivo del virus. El modelo de patosistema experimental utilizado en este trabajo está compuesto por el Virus del grabado del tabaco (TEV), aislado At17b (TEV-At17b), previamente adaptado por Agudelo-Romero et al. (2008)5 al ecotipo Ler-0 de A. thaliana, y por diferentes ecotipos de esta planta. En la primera parte de la Tesis, aplicando el método de hibridación de micromatrices de ARN, se caracterizó el transcriptoma de seis ecotipos de A. thaliana (Di-2, Ei-2, Ler-0 Oy-0, St-0 y Wt-1) tras ser infectados con el aislado TEV-At17b. El resultado de la infección se caracterizó fenotípicamente y también se determinó el patrón de expresión de genes alterados por la infección, así como las diferencias entre los distintos ecotipos. La respuesta era heterogénea, si bien los ecotipos se pudieron agrupar en dos grupos según su perfil transcriptómico. El propósito de la segunda parte del proyecto era explorar el efecto de la variabilidad genética intraespecífica del huésped en la dinámica de adaptación del virus. Con este fin, se realizó por triplicado un experimento de evolución con TEV-At17b en cinco ecotipos diferentes de A. thaliana (Di-2, Ei-2, Ler-0, St-0 y Wt-1). Después de la fase de evolución, se inocularon todos los ecotipos con todos los linajes virales evolucionados en un experimento de infección cruzada y se evaluaron la infectividad, la eficacia y la virulencia de cada combinación virus-ecotipo. Los resultados obtenidos se utilizaron para construir una red de infecciones. Las poblaciones de TEV, evolucionadas en cada uno de los cinco ecotipos de A. thaliana, aumentaron su eficacia, virulencia e infectividad en cada huésped de una manera compatible con un modelo gen-a-gen de interacciones parásito-huésped: los ecotipos difíciles de infectar fueron infectados por linajes generalistas, mientras que los ecotipos más susceptibles fueron infectados por cualquier linaje evolucionado. A continuación se caracterizaron los genomas completos de los virus evolucionados y se encontraron siete casos de mutaciones convergentes. En esta parte del proyecto, para obtener un mejor conocimiento de la base molecular de la interacción gen-a-gen, se generaron todas estas mutaciones de forma individual, así como combinaciones específicas, y se probaron sus efectos en plantas de los cinco ecotipos. Además, se evaluó la topografía del paisaje adaptativo subyacente. Usando el método de micromatrices de ARN, en el último capítulo de la Tesis se identificó la respuesta transcriptómica de cada ecotipo a la infección del virus evolucionado en simpatría. Los ecotipos del huésped se clasificaron en grupos de acuerdo a las similitudes en sus perfiles de expresión génica. Se evaluaron las categorías funcionales de los genes afectados por la infección y se determinaron las intersecciones y diferencias entre los ecotipos. A continuación, se caracterizó la respuesta transcriptómica del huésped ancestral Ler-0 a la infección con cada uno de los linajes evolucionados, cada uno adaptado a un ecotipo local diferente. Los cambios en las interacciones virus-huésped a nivel de expresión génica en Ler-0 eran homogéneos, pero mostraban cierta heterogeneidad en las categorías funcionales enriquecidas. Por último, se analizó la respuesta transcriptómica inducida por la cepa viral más especializada y la más generalista en los cinco ecotipos. Se encontró que el virus especialista inducía una respuesta más fuerte y más heterogénea en todos los ecotipos, mientras que el virus generalista alteraba la expresión génica de manera similar en todos los ecotipos del huésped. Por tanto, la estrategia adaptativa de un virus especialista parecía ser específica para su ecotipo simpátrico, pagando un coste de eficacia en su interacción con los huéspedes alopátricos. Por el contrario, la estrategia adaptativa del virus generalista consistía en la alteración de vías comunes para todos los ecotipos

Transcript Profiling of Different Arabidopsis thaliana Ecotypes in Response to Tobacco etch potyvirus Infection

The use of high-throughput transcript profiling techniques has opened the possibility of identifying, in a single experiment, multiple host mRNAs whose levels of accumulation are altered in response to virus infection. Several studies have used this approach to analyze the response of Arabidopsis thaliana to the infection by different RNA and DNA viruses. However, the possible differences in response of genetically heterogeneous ecotypes of the plant to the same virus have never been addressed before. Here we have used a strain of Tobacco etch potyvirus (TEV) experimentally adapted to A. thaliana ecotype Ler-0 and a set of seven plant ecotypes to tackle this question. Each ecotype was inoculated with the same amount of the virus and the outcome of infection characterized phenotypically (i.e., virus infectivity, accumulation, and symptoms development). Using commercial microarrays containing probes for more than 43,000 A. thaliana transcripts, we explored the effect of viral infection on the plant transcriptome. In general, we found that ecotypes differ in the way they perceive and respond to the virus. Some ecotypes developed strong symptoms and accumulated large amounts of viral genomes, while others only developed mild symptoms and accumulated less virus. At the transcriptomic level, ecotypes could be classified into two groups according to the particular genes whose expression was altered upon infection. Moreover, a functional enrichment analyses showed that the two groups differed in the nature of the altered biological processes. For the group constituted by ecotypes developing milder symptoms and allowing for lower virus accumulation, genes involved in abiotic stresses and in the construction of new tissues tend to be up-regulated. For those ecotypes in which infection was more severe and productive, defense genes tend to be up-regulated, deviating the necessary resources from building new tissues

Intra-specific variability and biological relevance of P3N-PIPO protein length in potyviruses

BackgroundPipo was recently described as a new ORF encoded within the genome of the Potyviridae family members (PNAS 105:5897–5902, 2008). It is embedded within the P3 cistron and is translated in the +2 reading frame relative to the potyviral long ORF as the P3N-PIPO fusion protein. In this work, we first collected pipo nucleotide sequences available for different isolates of 48 Potyvirus species. Second, to determine the biological implications of variation in pipo length, we measured infectivity, viral accumulation, cell-to-cell and systemic movements for two Turnip mosaic virus (TuMV) variants with pipo alleles of different length in three different susceptible host species, and tested for differences between the two variants.ResultsIn addition to inter-specific variation, there was high variation in the length of the PIPO protein among isolates within species (ranging from 1 to 89 amino acids). Furthermore, selection analyses on the P3 cistron did not account for the existence of stop codons in the pipo ORF, but showed that positive selection was significant in the overlapping region for Potato virus Y (PVY) and TuMV. In some cases, variability in length was associated with host species, geographic provenance and/or other strain features. We found significant empirical differences among the phenotypes associated with TuMV pipo alleles, though the magnitude and sign of the effects were host-dependent.ConclusionsThe combination of computational molecular evolution analyses and experiments stemming from these analyses provide clues about the selective pressures acting upon the different-length pipo alleles and show that variation in length may be maintained by host-driven selection

Experimental evolution of an emerging plant virus in host genotypes that differ in their susceptibility to infection

This study evaluates the extent to which genetic differences among host individuals from the same species condition the evolution of a plant RNA virus. We performed a threefold replicated evolution experiment in which Tobacco etch potyvirus isolate At17b (TEV-At17b), adapted to Arabidopsis thaliana ecotype Ler-0, was serially passaged in five genetically heterogeneous ecotypes of A. thaliana. After 15 passages we found that evolved viruses improved their fitness, showed higher infectivity and stronger virulence in their local host ecotypes. The genome of evolved lineages was sequenced and putative adaptive mutations identified. Host-driven convergent mutations have been identified. Evidences supported selection for increased translational efficiency. Next, we sought for the specificity of virus adaptation by infecting all five ecotypes with all 15 evolved virus populations. We found that some ecotypes were more permissive to infection than others, and that some evolved virus isolates were more specialist/generalist than others. The bipartite network linking ecotypes with evolved viruses was significantly nested but not modular, suggesting that hard-to-infect ecotypes were infected by generalist viruses whereas easy-to-infect ecotypes were infected by all viruses, as predicted by a gene-for-gene model of infection.We acknowledge grant BFU2012–30805 from the Spanish Ministerio de Economía y Competitividad to SFE. JMC was supported by a JAE-doc contract from CSIC. JH was supported by a pre-doctoral fellowship from Ministerio de Economía y CompetitividadPeer reviewe

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes

[EN] Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance and the rest of the EvolSysVir lab for fruitful discussions. This work was supported by grants PID2019-103998GB-I00 and PGC2018-101410-B-I00 (Agencia Estatal de Investigacion - FEDER) to S.F.E. and G.R., respectively, and PROMETEO/2019/012 (Generalitat Valenciana) to S.F.E.Martínez, F.; Carrasco, JL.; Toft, C.; Hillung, J.; Giménez-Santamarina, S.; Yenush, L.; Rodrigo Tarrega, G.... (2023). A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes. Communications Biology. 6(1):1-18. https://doi.org/10.1038/s42003-023-04427-81186

Temporal Dynamics of Intrahost Molecular Evolution for a Plant RNA Virus

[EN] 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.The authors thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance, our labmates for useful discussions and suggestions, and Dr Jose A. Daros for gifting us the pMTEV infectious clone. This work was supported by grants BFU2009-06993 and BFU2012-30805 from the Spanish Ministry of Economy and Competitiveness (MINECO), grant PROMETEOII/2014/021 from Generalitat Valenciana, and by the European Commission 7th Framework Programme (FP7-ICT-611640 FET Proactive: Evolving Living Technologies) EvoEvo project to S.F.E. J.M.C. was supported by a JAE-doc postdoctoral contract from CSIC. A.W. was supported by the EvoEvo project. J.H. was recipient of a predoctoral contract from MINECO. M.P.Z. was supported by a Juan de la Cierva postdoctoral contract from MINECO.Cuevas, JM.; Willemsen, A.; Hillung, J.; Zwart, MP.; Elena Fito, SF. (2015). Temporal Dynamics of Intrahost Molecular Evolution for a Plant RNA Virus. Molecular Biology and Evolution. 32(5):1132-1147. https://doi.org/10.1093/molbev/msv028S1132114732

Role of host genetic diversity for susceptibility-to-infection in the evolution of virulence of a plant virus

This article has been accepted for publication in Virus Evolution published by Oxford University Press.[EN] Predicting viral emergence is difficult due to the stochastic nature of the underlying processes and the many factors that govern pathogen evolution. Environmental factors affecting the host, the pathogen and the interaction between both are key in emergence. In particular, infectious disease dynamics are affected by spatiotemporal heterogeneity in their environments. A broad knowledge of these factors will allow better estimating where and when viral emergence is more likely to occur. Here, we investigate how the population structure for susceptibility-to-infection genes of the plant Arabidopsis thaliana shapes the evolution of Turnip mosaic virus (TuMV). For doing so we have evolved TuMV lineages in two radically different host population structures: (1) a metapopulation subdivided into six demes (subpopulations); each one being composed of individuals from only one of six possible A. thaliana ecotypes and (2) a well-mixed population constituted by equal number of plants from the same six A. thaliana ecotypes. These two populations were evolved for twelve serial passages. At the end of the experimental evolution, we found faster adaptation of TuMV to each ecotype in the metapopulation than in the well-mixed heterogeneous host populations. However, viruses evolved in well-mixed populations were more pathogenic and infectious than viruses evolved in the metapopulation. Furthermore, the viruses evolved in the demes showed stronger signatures of local specialization than viruses evolved in the well-mixed populations. 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Luria-Delbrück estimation of Turnip mosaic virus mutation rate in vivo

[EN] A potential drawback of recent antiviral therapies based on the transgenic expression of artificial microRNAs is the ease with which viruses may generate escape mutations. Using a variation of the classic Luria-Delbruck fluctuation assay, we estimated that the spontaneous mutation rate in the artificial microRNA (amiR) target of a plant virus was ca.6 x 10(-5) per replication event.This work was supported by grants BFU2009-06993 from the Spanish Ministerio de Ciencia e Innovación, RGP12/2008 from the Human Frontier Science Program Organization, and PROMETEO2010/019 from Generalitat Valenciana to S.F.E.; by CSIC grant 2010TW0015 to J.-A.D.; and by U.S. National Institutes of Health grants R01GM079843-01 and ARRA PDS#35063 and EC grant FP7231807 to P.J.G. F.M. was supported by a fellowship from Universidad Politénica de Valencia, J.H. was supported by a fellowship from the Spanish Ministerio de Ciencia e Innovación, and J.M.C. was contracted under the CSIC JAE-Doc program.De La Iglesia Jordán, F.; Martinez Garcia, F.; Hillung, J.; Cuevas Torrijos, JM.; Gerrish, PJ.; Daros Arnau, JA.; Elena Fito, SF. (2012). Luria-Delbrück estimation of Turnip mosaic virus mutation rate in vivo. Journal of Virology. 86(6):3386-3388. https://doi.org/10.1128/JVI.06909-11S3386338886

The transcriptomics of an experimentally evolved plant-virus interaction

[EN] Models of plant-virus interaction assume that the ability of a virus to infect a host genotype depends on the matching between virulence and resistance genes. Recently, we evolved tobacco etch potyvirus (TEV) lineages on different ecotypes of Arabidopsis thaliana, and found that some ecotypes selected for specialist viruses whereas others selected for generalists. Here we sought to evaluate the transcriptomic basis of such relationships. We have characterized the transcriptomic responses of five ecotypes infected with the ancestral and evolved viruses. Genes and functional categories differentially expressed by plants infected with local TEV isolates were identified, showing heterogeneous responses among ecotypes, although significant parallelism existed among lineages evolved in the same ecotype. Although genes involved in immune responses were altered upon infection, other functional groups were also pervasively over-represented, suggesting that plant resistance genes were not the only drivers of viral adaptation. Finally, the transcriptomic consequences of infection with the generalist and specialist lineages were compared. Whilst the generalist induced very similar perturbations in the transcriptomes of the different ecotypes, the perturbations induced by the specialist were divergent. Plant defense mechanisms were activated when the infecting virus was specialist but they were down-regulated when infecting with generalist.We thank Francisca de la Iglesia and Paula Agudo for excellent technical assistance and our labmates for useful discussions and suggestions. 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Evolutionary genomics of SARS-CoV-2: keys for viral success

Resumen del trabajo presentado al 44º Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Málaga del 6 al 9 de septiembre de 2022.Peer reviewe