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A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

ABSTRACT: The receptor-binding domain (RBD) of spike recognizing the receptor angiotensin-converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell-vesicle content-mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell-vesicle and cell-cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike-mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike-mediated SARS-CoV-2 entry.

SUBMITTER: Wu M

PROVIDER: S-EPMC10558659 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

Wu Mengdan M Li Wei W Lin Sheng S Fan Jiaqi J Cui Lele L Xiang Yijuan Y Li Kaiyu K Tang Linwei L Duan Yanping Y Chen Zimin Z Yang Fanli F Shui Weiwei W Lu Guangwen G Lai Ying Y

Advanced science (Weinheim, Baden-Wurttemberg, Germany) 20230817 28

The receptor-binding domain (RBD) of spike recognizing the receptor angiotensin-converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell-vesicle content-mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease st ...[more]

PMID: 37590389

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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the fusion machine for host cell entry. Still, the mechanism by which spike protein interacts with the target lipid membrane to facilitate membrane fusion during entry is not fully understood. Here, using steady-state membrane fusion and single-molecule fluorescence resonance energy transfer imaging of spike trimers on the surface of SARS-CoV-2 pseudovirion, we directly show that spike protein interacts with phosphatidylserine (PS) lipid in the target membrane for mediating fusion. We observed that the fusion peptide of the spike S2 domain interacts with the PS lipid of the target membrane. Low pH and Ca2+ trigger the spike conformational change and bring fusion peptide in close proximity to the PS lipid of the membrane. The binding of the spike with PS lipid of its viral membrane (cis interaction) impedes the fusion activation. PS on the target membrane promotes spike binding via trans interaction, prevents the cis interaction, and accelerates fusion. Sequestering or absence of PS lipid abrogates the spike-mediated fusion process and restricts SARS-CoV-2 infectivity. We found that PS-dependent interaction for fusion is conserved across all the SARS-CoV-2 spike variants of concern (D614G, Alpha, Beta, Delta, and Omicron). Our study suggests that PS lipid is indispensable for SARS-CoV-2 spike-mediated virus and target membrane fusion for entry, and restricting PS interaction with spike inhibits the SARS-CoV-2 spike-mediated entry. Therefore, PS is an important cofactor and acts as a molecular beacon in the target membrane for SARS-CoV-2 entry.ImportanceThe role of lipids in the host cell target membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is not clear. We do not know whether SARS-CoV-2 spike protein has any specificity in terms of lipid for membrane fusion reaction. Here, using in vitro reconstitution of membrane fusion assay and single-molecule fluorescence resonance energy transfer imaging of SARS-CoV-2 spike trimers on the surface of the virion, we have demonstrated that phosphatidylserine (PS) lipid plays a key role in SARS-CoV-2 spike-mediated membrane fusion reaction for entry. Membrane-externalized PS lipid strongly promotes spike-mediated membrane fusion and COVID-19 infection. Blocking externalized PS lipid with PS-binding protein or in the absence of PS, SARS-CoV-2 spike-mediated fusion is strongly inhibited. Therefore, PS is an important target for restricting viral entry and intervening spike, and PS interaction presents new targets for COVID-19 interventions.

Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a +sense single-strand RNA virus. The virus has four major surface proteins: spike (S), envelope (E), membrane (M), and nucleocapsid (N), respectively. The constitutive proteins present a high grade of symmetry. Identifying a binding site is difficult. The virion is approximately 50-200 nm in diameter. Angiotensin-converting enzyme 2 (ACE2) acts as the cell receptor for the virus. SARS-CoV-2 has an increased affinity to human ACE2 compared with the original SAR strain. Topological space, and its symmetry, is a critical component in molecular interactions. By exploring this space, a suitable ligand space can be characterized accordingly. A spike protein (S) computational model in a complex with ACE 2 was generated using silica methods. Topological spaces were probed using high computational throughput screening techniques to identify and characterize the topological space of both SARS and SARS-CoV-2 spike protein and its ligand space. In order to identify the symmetry clusters, computational analysis techniques, together with statistical analysis, were utilized. The computations are based on crystallographic protein data bank PDB-based models of constitutive proteins. Cartesian coordinates of component atoms and some cluster maps were generated and analyzed. Dihedral angles were used in order to compute a topological receptor space. This computational study uses a multimodal representation of spike protein interactions with some fragment proteins. The chemical space of the receptors (a dimensional volume) suggests the relevance of the receptor as a drug target. The spike protein S of SARS and SARS-CoV-2 is analyzed and compared. The results suggest a mirror symmetry of SARS and SARS-CoV-2 spike proteins. The results show thatSARS-CoV-2 space is variable and has a distinct topology. In conclusion, surface proteins grant virion variability and symmetry in interactions with a potential complementary target (protein, antibody, ligand). The mirror symmetry of dihedral angle clusters determines a high specificity of the receptor space.

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Dataset Information

A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

Publications

A Suitable Membrane Distance Regulated by the RBD_ACE2 Interaction is Critical for SARS-CoV-2 Spike-Mediated Viral Invasion.

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