SARS-CoV-2 Requires Cholesterol for Viral Entry and Pathological Syncytia Formation

Abstract

SummaryMany enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ∼6000 drugs and >30 spike variants. Together with cell biological and biophysical approaches, the screen reveals an essential role for membrane cholesterol in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins, and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.HighlightsCell-cell fusion at ACE2-spike clusters cause pathological syncytia in COVID-19Drug screen reveals critical role for membrane lipid composition in fusionSpike’s unusual membrane-proximal cysteines and aromatics are essential for fusionCholesterol tunes relative infectivity of SARS-CoV-2 viral particles

S-acylation controls SARS-Cov-2 membrane lipid organization and enhances infectivity

“…Using VLPs pseudotyped with WT or acylation deficient Spike, as well as SARS-CoV-2 virions produced from cells silenced for ZDHHC8/9/20 expression, we could show that acylation greatly enhances viral fusion and infectivity. Our findings are fully consistent and complementary to those by Brangwynne and co-workers who recently identified, through a screening approach, that cholesterol present in the membrane of VLPs is critical for Spike-mediated fusion (Sanders et al, 2020). Our study shows that S-acylation, and the ZDHHC20 enzyme in particular, constitutes a promising drug target for coronavirus infection.…”

“…Coronaviridae Spike proteins contain a conserved cysteine-rich stretch of amino acids in their cytosolic tail ( Figure 2A) (Gelhaus et al, 2014;Thorp et al, 2006). SARS-CoV-2 Spike has 10 cysteines within its first 20 cytosolic amino acids ( Figure 2A) and was recently highlighted as one of the most cysteine-rich proteins encoded by animal viruses (Sanders et al, 2020). To study the relative importance of these residues, we generated four mutants changing groups of 2-3 adjacent cysteines to alanine ( Figure 2A).…”

“…S-acylation, which consists in the covalent attachment of medium chain fatty acids (frequently palmitate, C16) to the sulphur atom of cytosolic cysteines, is very frequent in mammalian cells and considered a hallmark of viral envelope proteins Schmidt, 1982). S-acylation of Spike has been proposed to mediate its association to lipid microdomains, promote syncytia formation upon transfection into cells and reduce infectivity of MHV and of pseudotyped particles (McBride and Machamer, 2010;Nguyen et al, 2020;Sanders et al, 2020;Thorp et al, 2006).…”

Sequences in the cytoplasmic tail of SARS-CoV-2 Spike facilitate expression at the cell surface and syncytia formation

“…Although SARS-CoV-2 and most other coronaviruses do not bud from the cell surface, some S protein is found on the surface of infected cells. Consistent with this, infected cells have been observed to fuse with neighbouring cells to form large multinucleate cells or syncytia [15][16][17][18][19] . Thus, S must exit the ER to reach the ERGIC and later Golgi compartments, with some travelling all the way to the cell surface but the majority being retained at the site of viral budding.…”

“…It has recently been reported that syncytia formation by SARS-CoV-2 S can induce pyroptosis, and it has been proposed for other viral-mediated fusion events that cell death following syncytia formation can affect immune responses 60,61 . All this has led to an interest in the possibility that syncytia formation by SARS-CoV-2 is a potential target for therapeutic strategies 18,62 . Indeed, the process appears entirely dependent on the cell surface protease TMPRSS2 to make the activating S2' cleavage, whereas viral entry can be facilitated by either TMPRSS2 or lysosomal cathepsins 11,17 .…”