Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent behind the current COVID-19 pandemic having emerged in Wuhan China in late 2019 from a yet to be determined animal reservoir. SARS-CoV-2 B.1.1.7, a variant identified in the UK in late 2020, contains a higher than typical level of point mutants across its genome, including P681H in the spike S1/S2 cleavage site. Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from Wuhan-Hu1 and B.1.1.7 and observed no definitive difference in furin cleavage between Wuhan-Hu1 and B.1.1.7 in vitro. We performed functional assays using pseudo-typed particles harboring SARS-CoV-2 spike proteins and observed no significant differences between Wuhan-Hu1, Wuhan-Hu1 P681H or B.1.1.7 spike-carrying pseudo-typed particles in VeroE6 or Vero-TMPRSS2 cells, despite the spikes containing P681H being more efficiently cleaved. Likewise, we or show no differences in cell-cell fusion assays using the spike P681H-expressing cells. Our findings suggest that while the introduction of P681H in the SARS-CoV-2 B.1.1.7 variant may increase spike cleavage by furin-like proteases, this does not significantly impact viral entry or cell-cell spread. We consider that other factors are at play to account for the increased in transmission and disease severity attributed to this variant of concern (VOC).

Highlights SARS-CoV-2 B.1.1.7 P681H mutation in the spike is predicted to enhance viral infection P681H does not significantly impact furin cleavage, viral entry, or cell-cell spread Other mutations in the SARS-CoV-2 B.1.1.7 VOC may account for increased infection rates

The ability of SARS-CoV-2 to be primed for viral entry by the host cell protease furin has become one of the most investigated of the numerous transmission and pathogenicity features of the virus. Here, we analyzed the S1/S2 cleavage site (also called “furin cleavage site”) of the spike protein of SARS-CoV-2 B.1.529 (Omicron variant) in vitro, to assess the role of two key mutations (spike gene, N679K and P681H) compared to the ancestral B.1 virus. We observed significantly increased intrinsic cleavability with furin compared to an original B lineage virus (Wuhan-Hu-1) and two variants, B.1.1.7 (Alpha) and B.1.617 (Delta), that subsequently had wide circulation. Increased furin-mediated cleavage was attributed to the N679K mutation, which lies outside the conventional furin binding pocket. Our findings suggest that B.1.529 (Omicron variant) has gained genetic features linked to intrinsic furin cleavability, in line with its evolution within the population as the COVID-19 pandemic has proceeded.

The African continent is currently notable as 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 a Ugandan prison in July 2020, and then spilled into the general population adding 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 mutation 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 mutation may affect viral replication, transmissibility or pathogenic properties. Here, we performed assays using fluorogenic peptides mimicking the S1/S2 sequence from A.23.1 and observed significantly increased cleavability with furin, compared to sequences matching 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 in Vero-TMPRSS2 and Calu-3 cells, compared to Wuhan-Hu1, and a lowered infection in Vero E6 cells. However, these changes in infectivity were not reproduced in a P681R point mutant of Wuhan-Hu1 spike. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R mutation—which may affect viral infection and transmissibility—this mutation needs to occur on the background of other spike protein changes to enable its functional consequences.

During the course of the SARS-CoV-2 pandemic, viral variants have emerged that often contain notable mutations in the spike gene. Mutations that encode changes in the spike S1/S2 (furin) activation site have been considered especially impactful.