This study reveals a novel mechanism by which cholesterol promotes oligomerization of the spike protein and enhances viral membrane anchoring through interaction with the cysteine-rich region in its C-terminus, thereby driving SARS-CoV-2 membrane fusion, offering a potential target for antiviral intervention.
Literature Overview
The article "Molecular mechanism of cholesterol-dependent membrane fusion in SARS-CoV-2 entry," published in the journal Signal Transduction and Targeted Therapy, reviews and summarizes the critical regulatory role of cholesterol in the membrane fusion process mediated by the SARS-CoV-2 spike protein. Using a reconstituted in vitro vesicle-vesicle fusion system, single-vesicle imaging, and cell fusion assays, the study systematically demonstrates that cholesterol specifically binds to the cysteine-rich region (CRR) within the C-terminal intracellular domain of the spike protein, inducing its oligomerization into clusters, thereby significantly enhancing the efficiency of viral and host membrane anchoring and subsequently promoting membrane fusion and viral entry. This mechanism provides new insights into understanding coronavirus entry and suggests that targeting the cholesterol-spike interaction may represent a novel antiviral strategy.Background Knowledge
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped RNA virus whose entry into host cells relies on the binding of its spike glycoprotein to the host ACE2 receptor, triggering fusion between the viral and cellular membranes. Membrane fusion is a critical step in viral infection, regulated by multiple factors. Among these, the lipid composition of the membrane—particularly cholesterol—has been widely reported to influence the infection process of various enveloped viruses. Cholesterol not only modulates membrane fluidity and the formation of microdomains such as lipid rafts but can also directly participate in conformational changes of viral fusion proteins. Although previous studies have shown that SARS-CoV-2 infection is cholesterol-dependent, the precise molecular mechanisms remain unclear, especially regarding the dynamic assembly and membrane anchoring of the full-length spike protein. Moreover, while the C-terminal intracellular domain of the spike protein does not directly participate in receptor binding or fusion peptide insertion, its palmitoylation modifications may mediate interactions with the lipid environment. However, whether this domain acts as a cholesterol-sensing module to regulate the spatial organization and fusion efficiency of the spike protein has not been systematically elucidated. This study fills this gap by employing a multiscale experimental approach to reveal the pivotal functional role of cholesterol–spike CRR interaction in viral entry, providing a theoretical basis for developing novel antiviral strategies targeting viral entry.
Research Methods and Experiments
The research team established a physiologically relevant in vitro vesicle-vesicle fusion system, incorporating purified spike protein into liposomes (spike-vesicles) to mimic the viral membrane, while the extracellular domain of ACE2 was reconstituted into another set of liposomes labeled with fluorescent SRB (ACE2-vesicles) to simulate the host membrane. Membrane fusion efficiency was evaluated by monitoring fluorescence dequenching signals. Combined with single-vesicle imaging techniques, the independent effects of cholesterol on vesicle docking and fusion kinetics were further dissected. At the cellular level, HEK293T cells expressing spike protein and ACE2 were used to establish a cell–cell fusion model, and membrane cholesterol levels were modulated using MβCD or MβCD-CHO to assess syncytium formation. Pseudovirus infection assays with SARS-CoV-2 were performed to validate the impact of cholesterol on viral entry. To investigate the underlying molecular mechanism, structured illumination microscopy (SIM) was used to observe the spatial distribution of spike proteins on the cell membrane, and single-molecule photobleaching microscopy was employed to analyze their oligomeric states. Finally, C-terminal truncation mutants and CRR cysteine mutants were constructed to systematically evaluate functional deficits in cholesterol-dependent clustering and fusion.Key Conclusions and Perspectives
Research Significance and Prospects
This study is the first to systematically elucidate the molecular mechanism by which cholesterol interacts with the spike protein’s CRR region to drive its oligomerization and enhance viral membrane anchoring, revealing a novel function of the spike protein’s C-terminal domain as a 'cholesterol-sensing module.' This finding expands our understanding of coronavirus membrane fusion mechanisms and highlights how viruses exploit host lipid microenvironments to optimize their entry efficiency.
Targeting the cholesterol-spike interaction interface could become a novel intervention strategy to inhibit SARS-CoV-2 infection. For example, developing small molecules or peptide inhibitors to block CRR-cholesterol binding, or modulating membrane cholesterol distribution, may effectively inhibit viral entry. Furthermore, this mechanism may apply to other enveloped viruses dependent on lipid rafts or cholesterol, offering broad-spectrum antiviral potential. Future studies could further resolve the structural basis of the cholesterol-CRR interaction and explore its conservation and adaptive changes across viral variants.
Conclusion
This study integrates in vitro reconstitution systems, single-molecule imaging, and cellular functional assays to systematically reveal the critical regulatory role of cholesterol in the membrane fusion process mediated by the SARS-CoV-2 spike protein. The findings demonstrate that cholesterol does not merely passively modulate membrane physical properties, but instead actively drives the formation of higher-order oligomeric clusters by specifically binding to the cysteine-rich region (CRR) in the C-terminus of the spike protein, thereby significantly enhancing the anchoring efficiency between the viral and host membranes. This structural reorganization serves as a key prerequisite for efficient subsequent membrane fusion. Functional validation shows that disrupting CRR palmitoylation or deleting the C-terminal domain completely abolishes cholesterol’s promoting effect on membrane fusion. This mechanism provides new insights into how coronaviruses exploit host lipid environments to optimize their invasion strategies, and suggests that targeting the cholesterol-spike interaction interface may represent a potential approach for developing broad-spectrum antiviral drugs against enveloped viruses. The study not only deepens our understanding of SARS-CoV-2 entry mechanisms but also offers new molecular targets and theoretical foundations for antiviral interventions.
