In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes
العنوان: | In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes |
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المؤلفون: | Stéphane Bressanelli, Mohamed Debbah, Anja Böckmann, Johann Habersetzer, Marie-Laure Fogeron, Sonia Fieulaine |
المساهمون: | Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Interactions et mécanismes d’assemblage des protéines et des peptides (IMAPP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS) |
المصدر: | Protein Expression and Purification Protein Expression and Purification, Elsevier, 2020, ⟨10.1016/j.pep.2020.105694⟩ Protein Expression and Purification, 2020, ⟨10.1016/j.pep.2020.105694⟩ Protein Expression and Purification, 2020, pp.105694. ⟨10.1016/j.pep.2020.105694⟩ |
بيانات النشر: | Elsevier BV, 2020. |
سنة النشر: | 2020 |
مصطلحات موضوعية: | 0106 biological sciences, Polyproteins, [SDV]Life Sciences [q-bio], viruses, virus, Computational biology, Biology, maturation process, 01 natural sciences, Genome, Viral Proteins, 03 medical and health sciences, chemistry.chemical_compound, 010608 biotechnology, RNA polymerase, Humans, Gene, 030304 developmental biology, 0303 health sciences, Cell-Free System, Point mutation, Norovirus, RNA, RNA-Dependent RNA Polymerase, wheat-germ cell-free expression system, polyprotein, chemistry, Structural biology, Viral replication, Biotechnology |
الوصف: | International audience; Single-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic ma-turation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions. |
تدمد: | 1046-5928 1096-0279 |
DOI: | 10.1016/j.pep.2020.105694 |
DOI: | 10.1016/j.pep.2020.105694⟩ |
URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::700bd38ac09e4fa51cb1a287c7ea3ff1 https://doi.org/10.1016/j.pep.2020.105694 |
Rights: | OPEN |
رقم الانضمام: | edsair.doi.dedup.....700bd38ac09e4fa51cb1a287c7ea3ff1 |
قاعدة البيانات: | OpenAIRE |
تدمد: | 10465928 10960279 |
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DOI: | 10.1016/j.pep.2020.105694 |