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 additional spike mutations (R102I, L141F, E484K and P681R) to comprise lineage A.23.1 by September 2020-with this virus being designated a variant of interest (VOI) in Africa and with subsequent spread to 26 other countries. The P681R spike substitution of the A.23.1 VOI 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 appeared in B.1.617 variants, including B.1.617.2 (Delta). Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from A.23.1 and B.1.617.2 and observed significantly increased cleavability with furin, compared to sequences derived from the original Wuhan-Hu1 S1/S2. We performed functional infectivity assays using pseudotyped MLV particles harboring SARS-CoV-2 spike proteins and observed an increase in transduction for A.23.1-pseudotyped particles compared to Wuhan-Hu-1 in Vero-TMPRSS2 and Calu-3 cells (with a presumed "early" entry pathway), although lowered infection in Vero E6 cells (with a presumed "late" entry pathway). However, these changes in infectivity 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 is not sufficient and needs to occur on the background of other spike protein changes to enable its full functional consequences.