The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike (S) protein to mediate viral entry into host cells. Cleavage of the S protein at the S1/S2 and/or S2' site(s) is associated with viral entry, which can occur at either the cell plasma membrane (early pathway) or the endosomal membrane (late pathway), depending on the cell type. Previous studies show that SARS-CoV-2 has a unique insert at the S1/S2 site that can be cleaved by furin, which appears to expand viral tropism to cells with suitable protease and receptor expression. Here, we utilize viral pseudoparticles and protease inhibitors to study the impact of the S1/S2 cleavage on infectivity. Our results demonstrate that S1/S2 cleavage is essential for early pathway entry into Calu-3 cells, a model lung epithelial cell line, but not for late pathway entry into Vero E6 cells, a model cell line. The S1/S2 cleavage was found to be processed by other proteases beyond furin. Using bioinformatic tools, we also analyze the presence of a furin S1/S2 site in related CoVs and offer thoughts on the origin of the insertion of the furin-like cleavage site in SARS-CoV-2.

We analyzed the spike protein S1/S2 cleavage of selected strains of a prototype coronavirus, mouse hepatitis virus (MHV) by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of selected MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted results-but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

We have analyzed the spike protein S1/S2 cleavage site of selected strains of MHV by the cellular protease furin, in order to understand the structural requirements underlying the sequence selectivity of the scissile segment. The probability of cleavage of the various MHV strains was first evaluated from furin cleavage scores predicted by the ProP computer software, and then cleavage was measured experimentally with a fluorogenic peptide cleavage assay consisting of S1/S2 peptide mimics and purified furin. We found that in vitro cleavability varied across MHV strains in line with predicted resultsâ€"but with the notable exception of MHV-A59, which was not cleaved despite a high score predicted for its sequence. Using the known X-Ray structure of furin in complex with a substrate-like inhibitor as an initial structural reference, we carried out molecular dynamics (MD) simulations to learn the modes of binding of the peptides in the furin active site, and the suitability of the complex for initiation of the enzymatic cleavage. We thus identified the 3D structural requirements of the furin active site configuration that enable bound peptides to undergo cleavage, and the way in which the various strains tested experimentally are fulfilling these requirements. We find that despite some flexibility in the organization of the peptide bound to the active site of the enzyme, the presence of a histidine at P2 of MHV-A59 fails to properly orient the sidechain of His194 of the furin catalytic triad and therefore produces a distortion that renders the peptide/complex structural configuration in the active site incompatible with requirements for cleavage initiation. The Ser/Thr in P1 of MHV-2 and MHV-S has a similar effect of distorting the conformation of the furin active site residues produced by the elimination of the canonical salt-bridge formed by arginine in P1 position. This work informs a study of coronavirus infection and pathogenesis with respect to the function of the viral spike protein, and suggests an important process of viral adaptation and evolution within the spike S1/S2 structural loop.

Based on its predicted ability to affect transmissibility and pathogenesis, surveillance studies have highlighted the role of a specific mutation (P681R) in the S1/S2 furin cleavage site of the SARS-CoV-2 spike protein. Here we analyzed A.23.1, first identified in Uganda, as a P681R-containing virus several months prior to the emergence of B.1.617.2 (Delta variant). We performed assays using peptides mimicking the S1/S2 from A.23.1 and B.1.617 and observed significantly increased cleavability with furin compared to both an original B lineage (Wuhan-Hu1) and B.1.1.7 (Alpha variant). We also performed cell-cell fusion and functional infectivity assays using pseudotyped particles and observed an increase in activity for A.23.1 compared to an original B lineage spike. However, these changes in activity were not reproduced in the B lineage spike bearing only the P681R substitution. Our findings suggest that while A.23.1 has increased furin-mediated cleavage linked to the P681R substitution, this substitution needs to occur on the background of other spike protein changes to enable its functional consequences. IMPORTANCE 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. The S1/S2 change from proline to arginine at position 681 (P681R) first emerged in the A.23.1 variant in Uganda, and subsequently occurred in the more widely transmitted Delta variant. We show that the A.23.1 spike is more readily activated by the host cell protease furin, but that this is not reproduced in an original SARS-CoV-2 spike containing the P681R mutation. Changes to the S1/S2 (furin) activation site play a role in SARS-CoV-2 infection and spread, but successful viruses combine these mutations with other less well identified changes, occurring as part of natural selection.

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern first identified in the United Kingdom in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7, or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that, although the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage, this does not significantly impact viral entry or cell-cell spread.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent causing the COVID-19 pandemic. SARS-CoV-2 B.1.1.7 (Alpha), a WHO variant of concern (VOC) first identified in the UK in late 2020, contains several mutations including P681H in the spike S1/S2 cleavage site, which is predicted to increase cleavage by furin, potentially impacting the viral cell entry. Here, we studied the role of the P681H mutation in B.1.1.7 cell entry. We performed assays using fluorogenic peptides mimicking the Wuhan-Hu-1 and B.1.1.7 S1/S2 sequence and observed no significant difference in furin cleavage. Functional assays using pseudoparticles harboring SARS-CoV-2 spikes and cell-to-cell fusion assays demonstrated no differences between Wuhan-Hu-1, B.1.1.7 or a P681H point mutant. Likewise, we observed no differences in viral growth between USA-WA1/2020 and a B.1.1.7 isolate in cell culture. Our findings suggest that while the B.1.1.7 P681H mutation may slightly increase S1/S2 cleavage this does not significantly impact viral entry or cell-cell spread.

Feline Coronaviruses (FCoVs) commonly cause mild enteric infections in felines worldwide (termed Feline Enteric Coronavirus [FECV]), with around 12% developing into deadly Feline Infectious Peritonitis (FIP; Feline Infectious Peritonitis Virus [FIPV]). Genomic differences between FECV and FIPV have been reported, yet the putative genotypic basis of the highly pathogenic phenotype remains unclear. Here, we used state-of-the-art molecular evolutionary genetic statistical techniques to identify and compare differences in natural selection pressure between FECV and FIPV sequences, as well as to identify FIPV and FECV specific signals of positive selection. We analyzed full length FCoV protein coding genes thought to contain mutations associated with FIPV (Spike, ORF3abc, and ORF7ab). We identified two sites exhibiting differences in natural selection pressure between FECV and FIPV: one within the S1/S2 furin cleavage site, and the other within the fusion domain of Spike. We also found 15 sites subject to positive selection associated with FIPV within Spike, 11 of which have not previously been suggested as possibly relevant to FIP development. These sites fall within Spike protein subdomains that participate in host cell receptor interaction, immune evasion, tropism shifts, host cellular entry, and viral escape. There were 14 sites (12 novel) within Spike under positive selection associated with the FECV phenotype, almost exclusively within the S1/S2 furin cleavage site and adjacent C domain, along with a signal of relaxed selection in FIPV relative to FECV, suggesting that furin cleavage functionality may not be needed for FIPV. Positive selection inferred in ORF7b was associated with the FECV phenotype, and included 24 positively selected sites, while ORF7b had signals of relaxed selection in FIPV. We found evidence of positive selection in ORF3c in FCoV wide analyses, but no specific association with the FIPV or FECV phenotype. We hypothesize that some combination of mutations in FECV may contribute to FIP development, and that is unlikely to be one singular "switch" mutational event. This work expands our understanding of the complexities of FIP development and provides insights into how evolutionary forces may alter pathogenesis in coronavirus genomes.

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.

Coagulopathy is a significant aspect of morbidity in COVID-19 patients. The clotting cascade is propagated by a series of proteases, including factor Xa and thrombin. While certain host proteases, including TMPRSS2 and furin, are known to be important for cleavage activation of SARS-CoV-2 spike to promote viral entry in the respiratory tract, other proteases may also contribute. Using biochemical and cell-based assays, we demonstrate that factor Xa and thrombin can also directly cleave SARS-CoV-2 spike, enhancing infection at the stage of viral entry. Coagulation factors increased SARS-CoV-2 infection in human lung organoids. A drug-repurposing screen identified a subset of protease inhibitors that promiscuously inhibited spike cleavage by both transmembrane serine proteases and coagulation factors. The mechanism of the protease inhibitors nafamostat and camostat may extend beyond inhibition of TMPRSS2 to coagulation-induced spike cleavage. Anticoagulation is critical in the management of COVID-19, and early intervention could provide collateral benefit by suppressing SARS-CoV-2 viral entry. We propose a model of positive feedback whereby infection-induced hypercoagulation exacerbates SARS-CoV-2 infectivity.

Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2' fusion activation site-notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1' cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell-cell fusion and pseudotyped virus infectivity assays demonstrated that the S2' substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution.