Identification of a SARS-CoV-2 host metalloproteinase-dependent entry pathway differentially used by SARS-CoV-2 and variants of concern Alpha, Delta, and Omicron

Abstract

To infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinases activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

In silico analysis of mutations near S1/S2 cleavage site in SARS‐CoV‐2 spike protein reveals increased propensity of glycosylation in Omicron strain

“…Since furin can be found intracellularly in the trans‐Golgi network, extracellularly on the plasma membrane, or secreted into the extracellular matrix, the spike protein can be cleaved and, thus, primed for fusion before or after viral particle assembly (Figure 1). 11 Several proteases, such as TMPRSS2, MMP2/9, and neutrophil elastase, have been suggested to cleave the SARS‐CoV‐2 S1/S2 site as well 12,13 . Cleavage at the S2' site has been described to be cleaved by TMPRSS2 and related proteases.…”

“… 11 Several proteases, such as TMPRSS2, MMP2/9, and neutrophil elastase, have been suggested to cleave the SARS‐CoV‐2 S1/S2 site as well. 12 , 13 Cleavage at the S2' site has been described to be cleaved by TMPRSS2 and related proteases. VOC/VOI mutations in and around the known cleavage sites on the spike protein have been shown to modulate viral−host membrane fusion capacity and kinetics.…”

The P681H mutation in the Spike glycoprotein escapes IFITM restriction and is necessary for type I interferon resistance in the SARS-CoV-2 alpha variant

“…By contrast, PLVs displayed clear differences with the native virus – while all spikes were similarly expressed in the cell lysates, there were clear differences in the level of PLV incorporation of between PLVs (6D-6F), indicating that PLVs may not give a true reflection of the S conformation on native virions. Discrepancies between lentiviral vectors and virus S processing has also been recently suggested by the Cote group, and it is likely the cell type that the viruses and PLVs are produced in influences the observed S processing and may explain some of the differences in the literature [22, 37]. Given that the structural proteins E, M and N are known to regulate S retention, assembly, and glycosylation [38], we suggest that differences in S cleavage based solely on assays using spike-only PLVs should be interpreted with caution.…”

“…All of these variants have multiple changes in the S protein that could potentially affect the entry process (Figure 1). Of particular interest, the alpha, delta and omicron variants contain mutations in the polybasic cleavage site which have been postulated to enhance S cleavage: P681H in alpha and omicron, and P681R in delta [20][21][22]. We therefore compared the sensitivity of full-length S PLV entry of these VOCs in the presence of IFITM proteins.…”

Sphingosine kinases promote Ebola virus infection and can be targeted to inhibit filoviruses, coronaviruses, and arenaviruses using late endocytic trafficking to enter cells

“…As for SARS-CoV-1 and -2, the cellular localization of fusion depends on the expression and activity of cell surface proteases that are required for priming and/or triggering (33)(34)(35)(36). More specifically, SARS-CoV-1 can utilize surface serine proteases, while SARS-CoV-2 is more promiscuous in its protease usage, having been shown to utilize both surface serine proteases and metalloproteases for entry at the cell surface (33)(34)(35)(36)(37). In the absence of surface or extracellular proteases, the entry of SARS CoV-1 and -2 depends on internalization and trafficking to late endosomes containing cathepsin L (36,(38)(39)(40).…”