SARS-CoV-2 requires cholesterol for viral entry and pathological syncytia formation
Abstract
Many 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 quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially 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.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files with the exception of raw imaging data (>400,000 Nikon ND2 files), which is not feasible to post online given its massive size (>1.5 TB). This data is available from the lead contact upon request, assuming the interested party provides a server with sufficient storage capacity. Raw data (computed fusion scores) from the drug repurposing screen is available in Supplemental File 1; bioinformatics, Supplemental File 3.
Article and author information
Author details
Funding
National Institute of General Medical Sciences (GM095467)
- Bruce D Levy
National Heart, Lung, and Blood Institute (HL122531)
- Bruce D Levy
National Institute of General Medical Sciences (GM134949)
- Ilya Levental
National Institute of General Medical Sciences (GM124072)
- Ilya Levental
Howard Hughes Medical Institute
- Clifford P Brangwynne
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- William A Prinz, National Institutes of Health, United States
Ethics
Human subjects: Human pathology studies were performed with the approval of the Institutional Review Board at Brigham and Women's Hospital. Clinical autopsies with full anatomic dissection were performed on SARS-CoV-2 decedents by a board-certified anatomic pathologist (RFP) with appropriateinfectious precautions.
Version history
- Received: December 21, 2020
- Accepted: April 1, 2021
- Accepted Manuscript published: April 23, 2021 (version 1)
- Version of Record published: May 7, 2021 (version 2)
Copyright
© 2021, Sanders et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.
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Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here, we report that deletion of tricalbins (Tcb1, Tcb2, and Tcb3), tethering proteins at endoplasmic reticulum (ER)–plasma membrane (PM) and ER–Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine (PHS) accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus–vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous PHS and Tcb3-deleted cells, supporting that PHS transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids.