Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion

Abstract

The trimeric SARS-CoV-2 spike protein (S) is the sole viral protein responsible for both viral binding to a host cell and the membrane fusion event needed for cell entry. In addition to facilitating fusion needed for viral entry, S can also drive cell-cell fusion, a pathogenic effect observed in the lungs of SARS-CoV-2 infected patients. While several studies have investigated S requirements involved in viral particle entry, examination of S stability and factors involved in S cell-cell fusion remain limited. A furin cleavage site at the border between the S1 and S2 subunits (S1/S2) has been identified, along with putative cathepsin L and TMPRSS2 cleavage sites within S2. We demonstrate that S must be processed at the S1/S2 border in order to mediate cell-cell fusion, and that mutations at potential cleavage sites within the S2 subunit alter S processing at the S1/S2 border, thus preventing cell-cell fusion. We also identify residues within the internal fusion peptide and the cytoplasmic tail that modulate S-mediated cell-cell fusion. Additionally, we examined S stability and protein cleavage kinetics in a variety of mammalian cell lines, including a bat cell line related to the likely reservoir species for SARS-CoV-2, and provide evidence that proteolytic processing alters the stability of the S trimer. This work therefore offers insight into S stability, proteolytic processing, and factors that mediate S cell-cell fusion, all of which help give a more comprehensive understanding of this high profile therapeutic target.

Identifying Distinct Structural Features of the SARS-CoV-2 Spike Protein Fusion Domain Essential for Membrane Interaction

“…There have been a plethora of structural studies, both physical and computational, carried out on the SARS-CoV-2 spike protein since its discovery in December 2019. ,,,− While this has allowed the binding of the spike protein to the host cell receptor, ACE2, to be investigated extensively, the molecular mechanisms behind membrane fusion remain elusive. Imperative in this pursuit is the elucidation of the spike protein while associated with a lipid membrane; however, this poses its own challenges.…”

SARS-CoV-2 Delta (B.1.617.2) Variant: A Unique T478K Mutation in Receptor Binding Motif (RBM) of Spike Gene

“…Unlike SARS-CoV-2 α variant, 9 mutation sites of SARS-CoV-2 β variant are detected in subunit (S) 1 region, where the enzyme cleaves between N-terminal S1 (14–685 amino acid residues) region and C-terminal S2 (686–1,213 amino acid residues) region of spike extracellular protein ( Fig. 1B ) prior to SARS-CoV-2 entering a host cell ( 41 42 43 44 ). A single point mutation A701V presents in the S2 region that is distinct from SARS-CoV-2 α variant.…”

SARS‐CoV‐2 Alpha, Beta, and Delta variants display enhanced Spike‐mediated syncytia formation

“…We and others had previously shown that the S protein interacting with the ACE2 receptor induces cell–cell fusion (Buchrieser et␣al , 2020 ; Braga et␣al , 2021 ; Lin et␣al , 2021 ; Sanders et␣al , 2021 ; Zhang et␣al , 2021 ). The TMPRSS2 protease further augments cell–cell fusion (Buchrieser et␣al , 2020 ; Barrett et␣al , 2021 ; Hornich et␣al , 2021 ).…”