Date Published: February 14, 2019
Publisher: Public Library of Science
Author(s): Jocelyne Walter, Justine Charon, Yihua Hu, Joy Lachat, Thomas Leger, Guillaume Lafforgue, Amandine Barra, Thierry Michon, Ron Geller.
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.
Conformational intrinsic disorder is now recognized as a functional feature of a large number of proteins. The observation that protein or protein regions lacking stable and unique 3D conformations  carry important biological functions has revolutionized the old and well-established protein structure-function dogma. Since the birth in the early 2000s of the concept of protein intrinsic disorder, numerous studies have highlighted its ubiquitous and multi-functional nature, for review see [2,3]. The RNA virus proteome is particularly enriched in intrinsically disordered regions, IDRs [4,5]. Because of their structural plasticity, IDRs are multifunctional. Notably, many IDRs are involved in multi-partnership, thereby developing between the virus and its host, a complex interactome . A common trait of RNA viruses resides in their extraordinary ability to adapt to fast-changing environments, by creating a high degree of genetic diversity in viral populations. This genetic diversity both result from the unparalleled mutation rates observed in viruses, and their ability to evolve by maintaining mutations in populations, i.e mutational robustness [7,8]. Mutational robustness is defined as the ability of an organism to maintain its phenotype despite the disturbances caused by mutation accumulation. In case of viruses, this mutational robustness is the result of combined multi-scale processes including functional complementation between individual variants in the population (quasi-species) but also mutation buffering at the molecular level. Indeed, non-conservative mutations within viral proteins often lead to an increased instability, partly buffered by an enhanced chaperone assistance . Another and probably more frequent factor accounting for mutational robustness could reside in intrinsic structural properties of viral proteins . We previously proposed that IDRs could contribute to virus adaptive potential , and recently, we reported the first experimental evidence for the involvement of a viral protein intrinsically disordered region in the adaptation of a plant RNA virus to its host . An earlier study was aimed at comparing the structural features of 123 single-domain small-proteins from hypothermophylic bacteria, archaea, mesophilic eukaryota and prokaryota, and RNA or DNA viruses, whose crystal structures were available . It was concluded from this analysis that viral proteins and more particularly RNA virus proteins, display (i) higher stability upon simulations of mutation accumulation (lower ΔΔG per residues compared to the other groups of proteins) and (ii) lower inter-residues contact densities. This latter feature is a typical signature of intrinsic disorder. It has thus been proposed that the large intrinsic disorder content in viral protein could constitute a viral strategy to efficiently buffer mutation effects [13,14], strongly contrasting with non-additive/epistatic stability loss profile expected from ordered proteins as previously reported for a bacterial β-lactamase .
In order not to lose the high quality (size and diversity) of the libraries during their transfer from bacteria to yeasts, a cloning strategy was optimized. We used high efficiency Gateway recombination system, bacteria strains with maximum transformation efficiency and yeast mating that typically insures a better rate of cotransformed yeast. Corresponding mating efficiencies in yeast are provided in S2 Table. Typically, Libraries transformation in E.Coli produced an average of 3×104 individual clones. The higher cloning-mating efficiency in yeast (>106) insured that the original library complexity was conserved during the transfer between bacteria and yeast (see supporting information for details).