Spike Protein Cleavage-Activation Mediated by the SARS-CoV-2 P681R Mutation: A Case-Study From Its First Appearance in Variant of Interest (VOI) A.23.1 Identified in Uganda

The African continent like all other parts of the world with high infection/low vaccination rates can, and will, be a source of novel SARS-CoV-2 variants. The A.23 viral lineage, characterized by three spike mutations F157L, V367F and Q613H, was first identified in COVID-19 cases from a Ugandan prison in July 2020, and then was identified in the general population with the additional spike mutation P681R at the S1/S2 cleavage site to comprise lineage A.23.1 by September 2020 with subsequent spread to 26 other countries. The P681R spike substitution of A.23.1 is of note as it increases the number of basic residues in the sub-optimal SARS-CoV-2 spike protein furin cleavage site; as such, this substitution may affect viral replication, transmissibility, or pathogenic properties. The same P681R substitution has also subsequently appeared in B.1.617 variants, including B.1.617.2 (Delta). Here, we performed assays using fluorogenic peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin, compared to sequences derived from the original Wuhan-Hu1 S1/S2. We performed cell-cell fusion and functional infectivity assays using pseudotyped particles harboring SARS-CoV-2 spike proteins and observed an increase in transduction for A.23.1-pseudotyped particles compared to Wuhan-Hu-1. However, these changes in activity were not reproduced in the original Wuhan-Hu-1 spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution—which may affect viral infection and transmissibility—this substitution alone needs to occur on the background of other spike protein changes to enable its full functional consequences. Funding: This work was funded in part by the National Institute of Health research grant R01AI35270 (to GW and SD). We thank the global SARS-CoV-2 sequencing groups for their open and rapid sharing of sequence data and GISAID for providing an effective platform to make these data available. DLB, MVTP and MC were funded by the UK Medical Research Council (MRC/UK Research and Innovation) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement (grant agreement no. NC_PC_19060) and Wellcome Trust, UK FCDO—Wellcome Epidemic Preparedness—Coronavirus (grant agreement no. 220977/Z/20/Z). TT was supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. DGE-1650441 and the Samuel C. Fleming Family Graduate Fellowship. Declaration of Interests: The authors manifest no conflict of interest.