Integrative vectors for regulated expression of SARS-CoV-2 proteins implicated in RNA metabolism

Wellcome Open Research
S. BressonDavid Tollervey


Infection with SARS-CoV-2 is expected to result in substantial reorganization of host cell RNA metabolism. We identified 14 proteins that were predicted to interact with host RNAs or RNA binding proteins, based on published data for SARS-CoV and SARS-CoV-2. Here, we describe a series of affinity-tagged and codon-optimized expression constructs for each of these 14 proteins. Each viral gene was separately tagged at the N-terminus with Flag-His 8, the C-terminus with His 8-Flag, or left untagged. The resulting constructs were stably integrated into the HEK293 Flp-In T-REx genome. Each viral gene was expressed under the control of an inducible Tet-On promoter, allowing expression levels to be tuned to match physiological conditions during infection. Expression time courses were successfully generated for most of the fusion proteins and quantified by western blot. A few fusion proteins were poorly expressed, whereas others, including Nsp1, Nsp12, and N protein, were toxic unless care was taken to minimize background expression. All plasmids can be obtained from Addgene and cell lines are available. We anticipate that availability of these resources will facilitate a more detailed understanding of coronavirus molecular biology.


Mar 10, 2004·Proceedings of the National Academy of Sciences of the United States of America·Marie-Pierre EgloffBruno Canard
May 17, 2006·PLoS Biology·Grzegorz KudlaMaciej Zylicz
Jun 2, 2009·Proceedings of the National Academy of Sciences of the United States of America·Sander GrannemanDavid Tollervey
Jan 15, 2014·Antiviral Research·Chung-ke ChangTai-huang Huang
Jul 15, 2015·Proceedings of the National Academy of Sciences of the United States of America·Yuanyuan MaZihe Rao
Nov 2, 2016·Proceedings of the National Academy of Sciences of the United States of America·Tomonari MuramatsuShigeyuki Yokoyama
Nov 6, 2018·Nucleic Acids Research·Yasset Perez-RiverolJuan Antonio Vizcaíno
Apr 10, 2019·Molecular Systems Biology·Vadim ShchepachevDavid Tollervey
Apr 11, 2020·Cell Systems·Christine MordsteinGrzegorz Kudla
May 22, 2020·Nature·Hauke S HillenPatrick Cramer
Jun 9, 2020·BioRxiv : the Preprint Server for Biology·Thiruselvam ViswanathanYogesh K Gupta
Jun 12, 2020·Proceedings of the National Academy of Sciences of the United States of America·Ayal B GussowEugene V Koonin
Jun 13, 2020·Emerging Microbes & Infections·Chun-Kit YuenKin-Hang Kok
Jun 21, 2020·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Sean M Cascarina, Eric D Ross
Jun 27, 2020·BioRxiv : the Preprint Server for Biology·Jasmine CubukAlex S Holehouse
Jun 28, 2020·IScience·Dene R LittlerJamie Rossjohn

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