In a recent study published in the bioRxiv * prepress server, researchers at Weill Cornell Medical College evaluated the cleavability of the spike (S) protein of coronavirus 2 (SARS-CoV-2) strains of concern (VOC). severe and related acute respiratory syndrome. coronavirus by various host proteases.
Host proteases cut SARS-CoV-2 (and other coronaviruses) S at the border of S 1 and 2 subunits (S1 / S2), exposing peptides to the fusion of SARS-CoV-2 and cell membranes. host cell that facilitates SARS-CoV. -2 entry to the host. In a previous study, the authors showed that host proteases, such as clotting factors, cut down SARS-CoV-2 S and promote host invasion by SARS-CoV-2.
Study: Evolution of host protease interactions between SARS-CoV-2 variants of concern and related coronaviruses. NIAID image credit
About the study
In the present study, the researchers expanded their previous analysis by evaluating the S1 / S2 division of SARS-CoV-2 and other coronaviruses for host proteases such as furin, transmembrane protease serine 2 (TMPRSS2), thrombin, and coagulation Xa. They used fluorescence resonance energy transfer (FRET) assays to explore the impact of SARS-CoV-2 VOC mutations on the corresponding protein S division by host proteases. In addition, the division of S proteins from other coronaviruses by proteases was also evaluated.
Database subsamples such as Nextstrain and the Global Influenza All-Sharing Influenza (GISAID) initiative were examined for S 681 codon sequence divergence between SARS-CoV-2 VOCs. In particular, the team investigated the impact of mutations on SARS-CoV-2 variants in the S1 / S2 division by host proteases by comparing enzymatic kinetics. [maximum initial velocity (Vmax)] in substrates with P681 and P681H mutations corresponding to Wuhan-Hu-1 strain (ancestral) and VOC Alpha, respectively.
In addition, the activity of nafamostat in inhibiting the proteases involved in SARS-CoV-2 entry was evaluated. The effect of phosphorylation on host protease-mediated cleavage S1 / S2 on serine residues (Ser) located in the vicinity of the S1 / S2 cleavage site was determined by phosphorylated substrates corresponding to residues S689, S680, and S686.
In addition, the impact of post-translation modifications on S1 / S2 cleavability was assessed. Finally, the evolution of protease-mediated S1 / S2 cleavability through other related coronaviruses such as SARS-CoV, Middle East Respiratory Syndrome (MERS-CoV) coronavirus, the common cold virus HCoV-OC43), infectious bronchitis virus (IBV) – Beaudette and bat coronavirus (RatG13) were evaluated.
Results
The S 681 codon, which resides at the S1 / S2 cleavage site, was one of the highest entropy sites in the SARS-CoV-2 genome in the samples. The P681H and P681R mutations define the Alpha and Delta VOCs, respectively. Although the P681H mutation improved factor Xa activity, there were no changes in the cleavage activity of furin, thrombin, and TMPRSS2.
However, the P681R mutation improved the Vmax values of the S1 / S2 division of SARS-CoV-2 mediated by coagulation factor Xa and that of the S1 / S2 division mediated by furin by 65% and 99%, respectively. , compared to Wuhan-Soca Hu-1. In contrast, thrombin and TMPRSS2 showed reduced activity for the substrate expressing P681R.
Divergence of SARS-CoV-2 spike-681 sequences between worrying variants. (A) Outline of the SARS-CoV-2 ear protein, highlighting position 681 adjacent to the S1 / S2 site. Modified from (Kastenhuber et al., 2022). A subsampled collection of 3043 samples from GISAID between December 2109 and May 2022 was obtained and visualized using Nextstrain (Elbe and Buckland-Merrett, 2017; Hadfield et al., 2018). (B) Frequency of viral genomes sequenced with proline (black), histidine (red), or arginine (blue) at the 681 ear codon by sample collection date (C) Phylogenetic tree represented by Nextstrain The S681 genotype of each sample is indicated by proline (gray), histidine (red), or arginine (blue) The branches corresponding to the dominant variants of concern stand out in the outer ring.
Nafamostat inhibited the clotting factors, TMPRSS2 and other transmembrane serine proteases involved in the entry of SARS-CoV-2. While factor Xa showed elevated Vmax for variant substrates expressing P681Rv and P681H and mutations, factor Xa demonstrated equivalent nafamostat sensitivity to cleave P681Rv and P681H substrates. Ser 680 phosphorylation at the P6 position located upstream of the S1 / S2 cleavage site, fully completed furin-mediated cleavage, and moderately impacted S1 / S cleavage (30% to 50% inhibition) by factor Xa, thrombin, and TMPRSS2.
Ser 686 phosphorylation at the P1 position near the S1 / S2 cleavage site strongly inhibited the activity of all tested proteases. In contrast, phosphorylation of Ser 689 at the C4-terminal P4 position affected S1 / S2 cleavability so that TMPRSS2 and factor Xa were moderately inhibited, whereas thrombin was strongly inhibited and, conversely, observe an increase in furin-mediated cleavage.
Post-translation modifications by phosphorylation significantly affected the cleavability of S1 / S2. Protease susceptibility was different for all tested coronaviruses and only the SARS-CoV-2 cleavage site demonstrated division by the four tested proteases. Factor Xa demonstrated marked cleavability for the S1 / S2 cleavage sites of SARS-CoV-2 and HCoV-OC43.
Proteolytic fingerprint of various coronavirus lineages. (A) Phylogenic relationship of a coronavirus panel to the corresponding aligned S1 / S2 and S2 cleavage sites. Heat maps representing the initial rate V0 of cleavage of the indicated peptide substrates (rows) and concentrations (columns) by (B) factor Xa, (C) furin, (D) TMPRSS2, and (E) thrombin.
Furin showed efficacy against HCoV-OC43 strain S1 / S2-OC43 / Seattle, but not against HCoV-OC43 strain S1 / S2-OC43 / ATCC. In addition, furin efficiently split the S1 / S2 boundary and the S2 cleavage site of the IBV-Beaudette strain. Thrombin demonstrated the division of MERS, SARS, and SARS-CoV-2 S proteins, but did not substantially cleave RatG13.
TMPRSS2 cleaved S proteins (at cleavage sites S1 / S2 and S2) from several coronaviruses, although the efficiency of the cleavage was relatively low. In addition, the team observed the convergence of distant-related coronaviruses to achieve common interactions with host proteases, including coagulation factors. This indicates that the use of host proteases has probably been a pressure of selection through the evolution of coronaviruses.
Overall, the study findings showed that while SARS-CoV-2 VOCs had divergent interactions between their S1 / S2 cleavage sites and host proteases, a convergent evolution of cleavage mediated by host protease between different coronaviruses.
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