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. 2020 Oct 9;10:16944. doi: 10.1038/s41598-020-74101-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Phosphorylation at positions adjacent to the furin cleavage site inhibits the proteolytic cleavage in vitro. (a) The SARS-CoV2 S1/S2-GB1 reporter proteins were first phosphorylated with cyclin B-Cdk1 or PKA, followed by addition of furin. The furin activity was stopped at indicated time-points and the cleavage efficiency was analyzed by SDS-PAGE. (b) Plot showing the phosphorylation-dependent inhibition of furin activity on SARS-CoV2 S1/S2-GB1 reporter protein. Error bars show standard deviation. (c) Quantified furin cleavage profiles of the indicated SARS-CoV2 S1/S2-GB1 mutants without phosphorylation or with CDK- or PKA-mediated phosphorylation. Error bars show standard deviation. (d) Coomassie-stained SDS-PAGE gels showing the inhibitory effect of phosphorylation on furin-mediated cleavage of MERS-CoV S1/S2 with RRVR mutation. (e) Plot showing the relative abundance of cleaved MERS-CoV RRVR S1/S2-GB1 protein compared to the uncleaved form at t = 0. Error bars show standard deviation. Uncropped images are shown in Supplementary Fig. S4.