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Despite a rapid expansion in the number of documented viruses following the advent of metagenomic sequencing, the identification and annotation of highly divergent RNA viruses remain challenging, particularly from poorly characterized hosts and environmental samples. Protein structures are more conserved than primary sequence data, such that structure-based comparisons provide an opportunity to reveal the viral 'dusk matter': viral sequences with low, but detectable, levels of sequence identity to known viruses with available protein structures. Here, we present a new open computational resource-RdRp-scan-that contains a standardized bioinformatic toolkit to identify and annotate divergent RNA viruses in metagenomic sequence data based on the detection of RNA-dependent RNA polymerase (RdRp) sequences. By combining RdRp-specific hidden Markov models (HMMs) and structural comparisons, we show that RdRp-scan can efficiently detect RdRp sequences with identity levels as low as 10 per cent to those from known viruses and not identifiable using standard sequence-to-sequence comparisons. In addition, to facilitate the annotation and placement of newly detected and divergent virus-like sequences into the diversity of RNA viruses, RdRp-scan provides new custom and curated databases of viral RdRp sequences and core motifs, as well as pre-built RdRp multiple sequence alignments. In parallel, our analysis of the sequence diversity detected by the RdRp-scan revealed that while most of the taxonomically unassigned RdRps fell into pre-established clusters, some fell into potentially new orders of RNA viruses related to the Wolframvirales and Tolivirales. Finally, a survey of the conserved A, B, and C RdRp motifs within the RdRp-scan sequence database revealed additional variations of both sequence and position that might provide new insights into the structure, function, and evolution of viral polymerases.
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Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.
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Unicellular microalgae are of immense ecological importance with growing commercial potential in industries such as renewable energy, food, and pharmacology. Viral infections can have a profound impact on the growth and evolution of their hosts. However, very little is known of the diversity within, and the effect of, unicellular microalgal RNA viruses. In addition, identifying RNA viruses in these organisms that could have originated more than a billion years ago constitutes a robust data set to dissect molecular events and address fundamental questions in virus evolution. We assessed the diversity of RNA viruses in eight microalgal cultures, including representatives from the diatom, eustigmatophyte, dinoflagellate, red algae, and euglenid groups. Using metatranscriptomic sequencing combined with bioinformatic approaches optimized to detect highly divergent RNA viruses, we identified 10 RNA virus sequences, with nine constituting new viral species. Most of the newly identified RNA viruses belonged to the double-stranded Totiviridae, Endornaviridae, and Partitiviridae, greatly expanding the reported host range for these families. Two new species belonging to the single-stranded RNA viral clade Marnaviridae, commonly associated with microalgal hosts, were also identified. This study highlights that a substantial diversity of RNA viruses likely exists undetected within the unicellular microalgae. It also highlights the necessity for RNA viral characterization and for investigation of the effects of viral infections on microalgal physiology, biology, and growth, considering their environmental and industrial roles. IMPORTANCE Our knowledge of the diversity of RNA viruses infecting microbial algae-the microalgae-is minimal. However, describing the RNA viruses infecting these organisms is of primary importance at both the ecological and economic scales because of the fundamental roles these organisms play in aquatic environments and their growing value across a range of industrial fields. Using metatranscriptomic sequencing, we aimed to reveal the RNA viruses present in cultures of eight microalgae species belonging to the diatom, dinoflagellate, eustigmatophyte, rhodophyte, and euglena major clades of algae. Accordingly, we identified 10 new divergent RNA virus species belonging to RNA virus families as diverse as the double-stranded Totiviridae, Endornaviridae, and Partitiviridae and the single-stranded Marnaviridae. By expanding the known diversity of RNA viruses infecting unicellular eukaryotes, this study contributes to a better understanding of the early evolution of the virosphere and will inform the use of microalgae in industrial applications.
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Diatomeas , Dinoflagelados , Microalgas , Virus ARN , Diatomeas/genética , Dinoflagelados/genética , Microalgas/genética , Filogenia , Virus ARN/genética , Plantas , ARN , Genoma ViralRESUMEN
Intrinsically disordered regions (IDRs) are abundant in the proteome of RNA viruses. The multifunctional properties of these regions are widely documented and their structural flexibility is associated with the low constraint in their amino acid positions. Therefore, from an evolutionary stand point, these regions could have a greater propensity to accumulate non-synonymous mutations (NS) than highly structured regions (ORs, or 'ordered regions'). To address this hypothesis, we compared the distribution of non-synonymous mutations (NS), which we relate here to mutational robustness, in IDRs and ORs in the genome of potyviruses, a major genus of plant viruses. For this purpose, a simulation model was built and used to distinguish a possible selection phenomenon in the biological datasets from randomly generated mutations. We analyzed several short-term experimental evolution datasets. An analysis was also performed on the natural diversity of three different species of potyviruses reflecting their long-term evolution. We observed that the mutational robustness of IDRs is significantly higher than that of ORs. Moreover, the substitutions in the ORs are very constrained by the conservation of the physico-chemical properties of the amino acids. This feature is not found in the IDRs where the substitutions tend to be more random. This reflects the weak structural constraints in these regions, wherein an amino acid polymorphism is naturally conserved. In the course of evolution, potyvirus IDRs and ORs follow different evolutive paths with respect to their mutational robustness. These results have forced the authors to consider the hypothesis that IDRs and their associated amino acid polymorphism could constitute a potential adaptive reservoir.
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Potyvirus , Aminoácidos/genética , Electrólitos , Variación Genética , Potyvirus/genética , Proteoma/genéticaRESUMEN
Although water-borne viruses have important implications for the health of humans and other animals, little is known about the impact of human land use on viral diversity and evolution in water systems such as rivers. We used metatranscriptomic sequencing to compare the diversity and abundance of viruses at sampling sites along a single river in New Zealand that differed in human land-use impacts, ranging from pristine to urban. From this, we identified 504 putative virus species, of which 97 per cent were novel. Many of the novel viruses were highly divergent and likely included a new subfamily within the Parvoviridae. We identified at least sixty-three virus species that may infect vertebrates-most likely fish and water birds-from the Astroviridae, Birnaviridae, Parvoviridae, and Picornaviridae. No putative human viruses were detected. Importantly, we observed differences in the composition of viral communities at sites impacted by human land use (farming and urban) compared to native forest sites (pristine). At the viral species level, the urban sites had higher diversity (327 virus species) than the farming (n = 150) and pristine sites (n = 119), and more viruses were shared between the urban and farming sites (n = 76) than between the pristine and farming or urban sites (n = 24). The two farming sites had a lower viral abundance across all host types, while the pristine sites had a higher abundance of viruses associated with animals, plants, and fungi. We also identified viruses linked to agriculture and human impact at the river sampling sites in farming and urban areas that were not present at the native forest sites. Although based on a small sample size, our study suggests that human land use can impact viral communities in rivers, such that further work is needed to reduce the impact of intensive farming and urbanisation on water systems.
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Meta-transcriptomic next-generation sequencing has transformed virus discovery, dramatically expanding our knowledge of the known virosphere. Nevertheless, the use of meta-transcriptomics for virus discovery faces important challenges. As this technology becomes more widely adopted, the proportion of viral sequences in public databases with incorrect (e.g. mis-assignment of host) or limited information (e.g. lacking taxonomic classification) is likely to grow, limiting their utility in bioinformatic pipelines for virus discovery. In addition, we currently lack the bioinformatic tools that can accurately identify viruses showing little or no sequence similarity to database viruses or those that represent likely reagent contaminants. Herein, we outline some of the challenges to effective meta-transcriptomic virus discovery as well as their potential solutions.
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Transcriptoma/genética , Virología/métodos , Virus/clasificación , Virus/genética , Biología Computacional , Regulación Viral de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Virus/aislamiento & purificaciónRESUMEN
Remarkably little is known about the diversity and evolution of RNA viruses in unicellular eukaryotes. We screened a total of 570 transcriptomes from the Marine Microbial Eukaryote Transcriptome Sequencing Project that encompasses a wide diversity of microbial eukaryotes, including most major photosynthetic lineages (i.e. the microalgae). From this, we identified thirty new and divergent RNA virus species, occupying a range of phylogenetic positions within the overall diversity of RNA viruses. Approximately one-third of the newly described viruses comprised single-stranded positive-sense RNA viruses from the order Lenarviricota associated with fungi, plants, and protists, while another third were related to the order Ghabrivirales, including members of the protist and fungi-associated Totiviridae. Other viral species showed sequence similarity to positive-sense RNA viruses from the algae-associated Marnaviridae, the double-stranded RNA (ds-RNA) Partitiviridae, as well as tentative evidence for one negative-sense RNA virus related to the Qinviridae. Importantly, we were able to identify divergent RNA viruses from distant host taxa, revealing the ancestry of these viral families and greatly extending our knowledge of the RNA viromes of microalgal cultures. Both the limited number of viruses detected per sample and the low sequence identity to known RNA viruses imply that additional microalgal viruses exist that could not be detected at the current sequencing depth or were too divergent to be identified using sequence similarity. Together, these results highlight the need for further investigation of algal-associated RNA viruses as well as the development of new tools to identify RNA viruses that exhibit very high levels of sequence divergence.
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Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence.
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Chlorophyta/virología , Perfilación de la Expresión Génica , Filogenia , Virus ARN/clasificación , Evolución Molecular , Hongos/virología , Interacciones Microbiota-Huesped , Virus ARN/enzimología , ARN Polimerasa Dependiente del ARN , SimbiosisRESUMEN
The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg-eIF4E binding mechanism. Synthetic peptides encompassing the VPg88-120 central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg88-111 fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg112-120 region, which contains amino acids associated with resistance breakdown, is appended to VPg88-111, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg-eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.
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Factor 4E Eucariótico de Iniciación/química , Enfermedades de las Plantas/genética , Proteínas de Plantas/química , Potyvirus/genética , Secuencia de Aminoácidos/genética , Sitios de Unión/genética , Factor 4E Eucariótico de Iniciación/genética , Genoma Viral/genética , Interacciones Huésped-Patógeno/genética , Cinética , Defensa de la Planta contra la Herbivoria/genética , Enfermedades de las Plantas/virología , Proteínas de Plantas/genética , Potyvirus/química , Potyvirus/patogenicidad , Unión Proteica/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Eukaryotes of the genus Plasmodium cause malaria, a parasitic disease responsible for substantial morbidity and mortality in humans. Yet, the nature and abundance of any viruses carried by these divergent eukaryotic parasites is unknown. We investigated the Plasmodium virome by performing a meta-transcriptomic analysis of blood samples taken from patients suffering from malaria and infected with P. vivax, P. falciparum or P. knowlesi. This resulted in the identification of a narnavirus-like sequence, encoding an RNA polymerase and restricted to P. vivax samples, as well as an associated viral segment of unknown function. These data, confirmed by PCR, are indicative of a novel RNA virus that we term Matryoshka RNA virus 1 (MaRNAV-1) to reflect its analogy to a "Russian doll": a virus, infecting a parasite, infecting an animal. Additional screening revealed that MaRNAV-1 was abundant in geographically diverse P. vivax derived from humans and mosquitoes, strongly supporting its association with this parasite, and not in any of the other Plasmodium samples analyzed here nor Anopheles mosquitoes in the absence of Plasmodium. Notably, related bi-segmented narnavirus-like sequences (MaRNAV-2) were retrieved from Australian birds infected with a Leucocytozoon-a genus of eukaryotic parasites that group with Plasmodium in the Apicomplexa subclass hematozoa. Together, these data support the establishment of two new phylogenetically divergent and genomically distinct viral species associated with protists, including the first virus likely infecting Plasmodium parasites. As well as broadening our understanding of the diversity and evolutionary history of the eukaryotic virosphere, the restriction to P. vivax may be of importance in understanding P. vivax-specific biology in humans and mosquitoes, and how viral co-infection might alter host responses at each stage of the P. vivax life-cycle.
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Malaria Vivax/parasitología , Parásitos/genética , Plasmodium vivax/genética , Plasmodium/genética , Virus ARN/genética , Animales , Anopheles/parasitología , Enfermedades de las Aves , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/genéticaRESUMEN
Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.
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Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Lactuca/metabolismo , Lactuca/virología , Proteínas de Plantas/metabolismo , Potyvirus/fisiología , Proteínas Virales/metabolismo , Cromatografía en Gel , Factor 4E Eucariótico de Iniciación/química , Interacciones Huésped-Patógeno , Hidrodinámica , Proteínas Intrínsecamente Desordenadas/química , Lactuca/química , Enfermedades de las Plantas/virología , Proteínas de Plantas/química , Potyvirus/química , Proteínas Virales/químicaRESUMEN
Conformational intrinsic disorder is a feature present in many virus proteins. Intrinsically disordered regions (IDRs) have weaker structural requirement than ordered regions and mutations in IDRs could have a lower impact on the virus fitness. This could favor its exploration of adaptive solutions. The potyviral protein VPg contains IDRs with determinants for adaptation to its host plant. To experimentally assess whether IDRs are more resistant to mutations than ordered regions, the biologically relevant interaction between mutant libraries of both VPg and the eukaryotic translation initiation factor 4E (eIF4E) and their respective wild type partner was examined using yeast two hybrid assay. Our data shows that VPg is significantly more robust to mutations than eIF4E and as such belongs to a particular class of intrinsically disordered proteins. This result is discussed from the standpoint of IDRs involvement in the virus adaptive processes.
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Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas de Plantas/metabolismo , Potyvirus/metabolismo , Proteínas Virales/metabolismo , Capsicum/genética , Capsicum/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Interacciones Huésped-Patógeno/genética , Proteínas Intrínsecamente Desordenadas/genética , Mutación/genética , Proteínas de Plantas/genética , Potyvirus/genética , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genéticaRESUMEN
The threat of bacteria resistant to multiple antibiotics poses a major public health problem requiring immediate and coordinated action worldwide. While infectious pathogens have become increasingly resistant to commercially available drugs, antibiotic discovery programs in major pharmaceutical companies have produced no new antibiotic scaffolds in 40 years. As a result, new strategies must be sought to obtain a steady supply of novel scaffolds capable of countering the spread of resistance. The bacterial ribosome is a major target for antimicrobials and is inhibited by more than half of the antibiotics used today. Recent studies showing that the ribosome is a target for several classes of ribosomally synthesized antimicrobial peptides point to ribosome-targeting peptides as a promising source of antibiotic scaffolds. In this Perspective, we revisit the current paradigm of antibiotic discovery by proposing that the bacterial ribosome can be used both as a target and as a tool for the production and selection of peptide-based antimicrobials. Turning the ribosome into a high-throughput platform for the directed evolution of peptide-based antibiotics could be achieved in different ways. One possibility would be to use a combination of state-of-the-art microfluidics and genetic reprogramming techniques, which we will review briefly. If it is successful, this strategy has the potential to produce new classes of antibiotics for treating multi-drug-resistant pathogens.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Péptidos/metabolismo , Péptidos/farmacología , Ribosomas/efectos de los fármacos , Antibacterianos/química , Bacterias/genética , Bacterias/metabolismo , Evolución Molecular Dirigida/métodos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Estudios de Asociación Genética , Ensayos Analíticos de Alto Rendimiento/métodos , Biblioteca de Péptidos , Péptidos/química , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Intrinsic disorder (ID) in proteins is defined as a lack of stable structure in physiological conditions. Intrinsically disordered regions (IDRs) are highly abundant in some RNA virus proteomes. Low topological constraints exerted on IDRs are expected to buffer the effect of numerous deleterious mutations and could be related to the remarkable adaptive potential of RNA viruses to overcome resistance of their host. To experimentally test this hypothesis in a natural pathosystem, a set of four variants of Potato virus Y (PVY; Potyvirus genus) containing various ID degrees in the Viral genome-linked (VPg) protein, a key determinant of potyvirus adaptation, was designed. To estimate the ID contribution to the VPg-based PVY adaptation, the adaptive ability of the four PVY variants was monitored in the pepper host (Capsicum annuum) carrying a recessive resistance gene. Intriguingly, the two mutants with the highest ID content displayed a significantly higher ability to restore infection in the resistant host, whereas the less intrinsically disordered mutant was unable to restore infection. The role of ID on virus adaptation may be due either to a larger exploration of evolutionary pathways or the minimization of fitness penalty caused by resistance-breaking mutations. This pioneering study strongly suggests the positive impact of ID in an RNA virus adaptive capacity.
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Adaptación Fisiológica/genética , Potyvirus/genética , Ribonucleoproteínas/genética , Proteínas no Estructurales Virales/genética , Aclimatación/genética , Evolución Biológica , Capsicum/virología , Evolución Molecular , Genoma Viral , Mutación/genética , Proyectos Piloto , Estabilidad Proteica , Proteoma , ARN/genética , Ribonucleoproteínas/fisiología , Proteínas no Estructurales Virales/fisiologíaRESUMEN
Reproductive development of grapevine and berry composition are both strongly influenced by temperature. To date, the molecular mechanisms involved in grapevine berries response to high temperatures are poorly understood. Unlike recent data that addressed the effects on berry development of elevated temperatures applied at the whole plant level, the present work particularly focuses on the fruit responses triggered by direct exposure to heat treatment (HT). In the context of climate change, this work focusing on temperature effect at the microclimate level is of particular interest as it can help to better understand the consequences of leaf removal (a common viticultural practice) on berry development. HT (+ 8°C) was locally applied to clusters from Cabernet Sauvignon fruiting cuttings at three different developmental stages (middle green, veraison and middle ripening). Samples were collected 1, 7, and 14 days after treatment and used for metabolic and transcriptomic analyses. The results showed dramatic and specific biochemical and transcriptomic changes in heat exposed berries, depending on the developmental stage and the stress duration. When applied at the herbaceous stage, HT delayed the onset of veraison. Heating also strongly altered the berry concentration of amino acids and organic acids (e.g., phenylalanine, γ-aminobutyric acid and malate) and decreased the anthocyanin content at maturity. These physiological alterations could be partly explained by the deep remodeling of transcriptome in heated berries. More than 7000 genes were deregulated in at least one of the nine experimental conditions. The most affected processes belong to the categories "stress responses," "protein metabolism" and "secondary metabolism," highlighting the intrinsic capacity of grape berries to perceive HT and to build adaptive responses. Additionally, important changes in processes related to "transport," "hormone" and "cell wall" might contribute to the postponing of veraison. Finally, opposite effects depending on heating duration were observed for genes encoding enzymes of the general phenylpropanoid pathway, suggesting that the HT-induced decrease in anthocyanin content may result from a combination of transcript abundance and product degradation.
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Within proteins, intrinsically disordered regions (IDRs) are devoid of stable secondary and tertiary structures under physiological conditions and rather exist as dynamic ensembles of inter-converting conformers. Although ubiquitous in all domains of life, the intrinsic disorder content is highly variable in viral genomes. Over the years, functional annotations of disordered regions at the scale of the whole proteome have been conducted for several animal viruses. But to date, similar studies applied to plant viruses are still missing. Based on disorder prediction tools combined with annotation programs and evolutionary studies, we analyzed the intrinsic disorder content in Potyvirus, using a 10-species dataset representative of this genus diversity. In this paper, we revealed that: (i) the Potyvirus proteome displays high disorder content, (ii) disorder is conserved during Potyvirus evolution, suggesting a functional advantage of IDRs, (iii) IDRs evolve faster than ordered regions, and (iv) IDRs may be associated with major biological functions required for the Potyvirus cycle. Notably, the proteins P1, Coat protein (CP) and Viral genome-linked protein (VPg) display a high content of conserved disorder, enriched in specific motifs mimicking eukaryotic functional modules and suggesting strategies of host machinery hijacking. In these three proteins, IDRs are particularly conserved despite their high amino acid polymorphism, indicating a link to adaptive processes. Through this comprehensive study, we further investigate the biological relevance of intrinsic disorder in Potyvirus biology and we propose a functional annotation of potyviral proteome IDRs.
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Potyvirus , Proteoma/química , Proteínas Virales/química , Proteínas Intrínsecamente Desordenadas , Modelos Moleculares , Anotación de Secuencia Molecular , Filogenia , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteolisis , Proteoma/fisiología , Análisis de Secuencia de Proteína , Proteínas Virales/fisiologíaRESUMEN
Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Grapevine is the most cultivated fruit plant throughout the world, and grapes are used for wine production and human consumption. The molecular mechanisms involved in grapevine tolerance to high temperature, especially at the fruit level, are poorly understood. To better characterize the sensitivity of berries to the microenvironment, high temperature conditions were locally applied to Vitis vinifera Cabernet Sauvignon clusters. Two genes, VvGOLS1 and VvHsfA2, up-regulated by this treatment, were identified and further characterized. The expression profile of VvGOLS1 correlated positively with galactinol accumulation in heat-stressed berries. However, no galactinol derivatives, such as raffinose and stachyose, accumulated upon heat stress. Heterologous expression of VvGOLS1 in Escherichia coli showed that it encodes a functional galactinol synthase. Transient expression assays showed that the heat stress factor VvHsfA2 transactivates the promoter of VvGOLS1 in a heat stress-dependent manner. Taken together, our results highlight the intrinsic capacity of grape berries to perceive heat stress and to initiate adaptive responses, suggesting that galactinol may play a signaling role in these responses.