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Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein.

ABSTRACT: RNA virus high mutation rate is a double-edged sword. At the one side, most mutations jeopardize proteins functions; at the other side, mutations are needed to fuel adaptation. The relevant question then is the ratio between beneficial and deleterious mutations. To evaluate this ratio, we created a mutant library of the 6K2 gene of tobacco etch potyvirus that contains every possible single-nucleotide substitution. 6K2 protein anchors the virus replication complex to the network of endoplasmic reticulum membranes. The library was inoculated into the natural host Nicotiana tabacum, allowing competition among all these mutants and selection of those that are potentially viable. We identified 11 nonsynonymous mutations that remain in the viral population at measurable frequencies and evaluated their fitness. Some had fitness values higher than the wild-type and some were deleterious. The effect of these mutations in the structure, transmembrane properties, and function of 6K2 was evaluated in silico. In parallel, the effect of these mutations in infectivity, virus accumulation, symptoms development, and subcellular localization was evaluated in the natural host. The ?-helix H1 in the N-terminal part of 6K2 turned out to be under purifying selection, while most observed mutations affect the link between transmembrane ?-helices H2 and H3, fusing them into a longer helix and increasing its rigidity. In general, these changes are associated with higher within-host fitness and development of milder or no symptoms. This finding suggests that in nature selection upon 6K2 may result from a tradeoff between within-host accumulation and severity of symptoms.

SUBMITTER: Gonzalez R

PROVIDER: S-EPMC6482416 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein.

González Rubén R Wu Beilei B Li Xianghua X Martínez Fernando F Elena Santiago F SF

Genome biology and evolution 20190401 4

RNA virus high mutation rate is a double-edged sword. At the one side, most mutations jeopardize proteins functions; at the other side, mutations are needed to fuel adaptation. The relevant question then is the ratio between beneficial and deleterious mutations. To evaluate this ratio, we created a mutant library of the 6K2 gene of tobacco etch potyvirus that contains every possible single-nucleotide substitution. 6K2 protein anchors the virus replication complex to the network of endoplasmic re ...[more]

PMID: 30918938

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Project description:BackgroundThe genomes of plant viruses have limited coding capacity, and to complete their infectious cycles, viral factors must target, direct or indirectly, many host elements. However, the interaction networks between viruses and host factors are poorly understood. The genus Potyvirus is the largest group of plus-strand RNA viruses infecting plants. Potyviral nuclear inclusion a (NIa) plays many roles during infection. NIa is a polyprotein consisting of two domains, viral protein genome-linked (VPg) and protease (NIaPro), separated by an inefficiently utilized self-proteolytic site. To gain insights about the interaction between potyviral NIa and the host cell during infection, we constructed Tobacco etch virus (TEV, genus Potyvirus) infectious clones in which the VPg or the NIaPro domains of NIa were tagged with the affinity polypeptide Twin-Strep-tag and identified the host proteins targeted by the viral proteins by affinity purification followed by mass spectrometry analysis (AP-MS).ResultsWe identified 232 different Arabidopsis thaliana proteins forming part of complexes in which TEV NIa products were also involved. VPg and NIaPro specifically targeted 89 and 76 of these proteins, respectively, whereas 67 proteins were targeted by both domains and considered full-length NIa targets. Taking advantage of the currently known A. thaliana interactome, we constructed a protein interaction network between TEV NIa domains and 516 host proteins. The most connected elements specifically targeted by VPg were G-box regulating factor 6 and mitochondrial ATP synthase δ subunit; those specifically targeted by NIaPro were plasma membrane aquaporin PIP2;7 and actin 7, whereas those targeted by full-length NIa were heat shock protein 70-1 and photosystem protein LHCA3. Moreover, a contextualization in the global A. thaliana interactome showed that NIa targets are not more connected with other host proteins than expected by chance, but are in a position that allows them to connect with other host proteins in shorter paths. Further analysis of NIa-targeted host proteins revealed that they are mainly involved in response to stress, metabolism, photosynthesis, and localization. Many of these proteins are connected with the phytohormone ethylene.ConclusionsPotyviral NIa targets many host elements during infection, establishing a network in which information is efficiently transmitted.

Dataset Information

Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein.

Publications

Mutagenesis Scanning Uncovers Evolutionary Constraints on Tobacco Etch Potyvirus Membrane-Associated 6K2 Protein.

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