SARS‐CoV‐2 is the coronavirus responsible for the COVID‐19 pandemic. Proteases are central to the infection process of SARS‐CoV‐2. Cleavage of the spike protein on the virus's capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS‐CoV‐2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS‐CoV‐2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID‐19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.
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Analysis of the molecular determinants for furin cleavage of the spike protein S1/S2 site in defined strains of the prototype coronavirus murine hepatitis virus (MHV)
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.
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- Award ID(s):
- 1740990
- NSF-PAR ID:
- 10481486
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Virus Research
- Volume:
- 340
- Issue:
- C
- ISSN:
- 0168-1702
- Page Range / eLocation ID:
- 199283
- Subject(s) / Keyword(s):
- ["furin\nmouse hepatitis virus\ncoronavirus\nspike protein\nstructural criteria for spike cleavage by furin"]
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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