Unveiling the First Step of Viral Fusion: Scientists Capture Early Fusion Intermediate of ACE2-Mediated Coronavirus

Since its outbreak in late 2019, SARS-CoV-2 has profoundly impacted global public health. The virus infects host cells by binding its Spike protein (S protein) to the ACE2 (Angiotensin-Converting Enzyme 2) receptor, a process involving complex membrane fusion mechanisms. This process includes initial ACE2 receptor binding, S1 subunit shedding, and the formation of a six-helix bundle (6-HB) structure, facilitating viral and host cell membrane fusion. However, the structural and functional characteristics of the early fusion intermediate between ACE2 binding and S1 shedding remain unclear, representing a critical knowledge gap for antiviral drug development. Addressing this, the research group led by Lixiao Xing at Fudan University published a study in CELL titled "Early Fusion Intermediate of ACE2-Using Coronavirus Spike Acting as an Antiviral Target." Using structural biology, cell experiments, and animal models, the study systematically analyzes the early fusion intermediate of ACE2-dependent coronavirus S protein, revealing its potential as an antiviral target and providing crucial insights for novel antiviral strategies.

Structural Analysis of the Early Fusion Intermediate of the S Protein

The research team utilized cryo-electron microscopy (Cryo-EM) to resolve the structure of the ACE2-induced early fusion intermediate (E-FIC-S-ACE2 complex) of the S protein. They discovered that the S protein adopts a unique conformation upon ACE2 induction, exposing the HR1 domain, which plays a critical role in membrane fusion and serves as a potential antiviral target. The exposure of HR1 provides a structural basis for designing inhibitors targeting the fusion process.

Development and Specificity Validation of the Novel Antiviral Candidate ALSE

Based on this finding, the team designed a protein molecule, ALSE, targeting the HR1 domain to specifically block the fusion process. In vitro experiments demonstrated that ALSE exhibits significant inhibitory activity against ACE2-dependent coronaviruses but shows no effect on non-ACE2-dependent viruses, indicating high specificity. This suggests that E-FIC is a unique target for SARS-CoV-2 infection, and ALSE effectively prevents viral entry by targeting HR1. Fluorescence focus formation assays further confirmed ALSE's inhibitory and viral particle inactivation activities against authentic coronaviruses, demonstrating its potential as a broad-spectrum antiviral.

Antiviral Efficacy of ALSE in Animal Models

• To evaluate ALSE's in vivo efficacy, the study assessed inhaled ALSE administration in transgenic mice expressing human ACE2, targeting SARS-CoV-2 BA.2.5 variant infection. Results showed that the ALSE-treated group had significantly reduced lung viral loads compared to the control group, with no significant expression of pro-inflammatory cytokines observed.
• H&E staining analysis revealed no significant tissue damage in organs such as the lung, spleen, or kidney, indicating good safety. Fluorescence imaging further showed that ALSE has a long half-life in the lungs, suggesting suitability for inhaled administration to achieve high local concentrations. Additionally, ALSE exhibited low immunogenicity in vivo, inducing no significant antibody responses, indicating excellent biosafety.

In-Depth Exploration of the Fusion Mechanism

• The study further analyzed the impact of the S2 subunit cleavage site (S2' site) on ACE2-induced HR1 collapse, revealing the dynamic molecular mechanisms of S protein fusion conformational transitions. It confirmed that S2' site cleavage is a critical step for HR1 exposure and membrane fusion. Experiments with EK1 peptide and anti-HR1 antibodies validated the dynamic exposure of HR1 during fusion, further supporting its feasibility as an antiviral target.

Despite significant breakthroughs, the study has limitations. The S protein constructs used lacked the transmembrane region, limiting simulation of the complete fusion process. Additionally, high-resolution structures of the E-FIC-PG complex were not obtained, and validation in non-human primate models is still needed, necessitating further optimization of experimental designs.

Introduction to the ACE2 Target

ACE2 (Angiotensin-Converting Enzyme 2) is a membrane-bound metalloproteinase widely expressed in human tissues such as the lungs, kidneys, and heart. Its primary function is to regulate the renin-angiotensin system (RAS) by converting angiotensin II (Ang II) to angiotensin 1-7 (Ang1-7), thereby controlling blood pressure, inflammation, and tissue fibrosis. In coronavirus infections, ACE2 serves as the receptor for SARS-CoV-2 and other viruses, mediating viral entry into host cells via binding to the S protein's receptor-binding domain (RBD). This binding triggers conformational changes that initiate the membrane fusion process, a critical step in viral infection.

In recent years, ACE2 has garnered significant attention as a target for anti-coronavirus drug development. Research focuses on the following directions:

       1.   Neutralizing Antibodies and Soluble ACE2 (From Blocking to Decoying): Developing neutralizing antibodies targeting the S protein-ACE2 interaction (e.g., Regeneron and AstraZeneca's anti-S protein bispecific antibodies) to block viral entry, or using soluble ACE2 proteins as decoys to bind viruses and neutralize their infectivity (e.g., Apeiron Biologics' APN01 project).

       2.   Small Molecule Inhibitors: Small molecules offer advantages in oral bioavailability and cost. Teams have screened natural products and drug libraries for molecules that block S protein-ACE2 interactions (e.g., Exscientia's AI-screened EXS4318, now in preclinical studies), showing promising binding inhibition.

       3.   Fusion Inhibitors: As described in this study, inhibitors targeting the S protein's fusion intermediate (e.g., HR1) provide a novel approach to antiviral drug design.

       4.   ACE2 Modulation Therapies: Exploring the upregulation or downregulation of ACE2 expression to balance its dual roles in viral infection and physiological regulation. However, ACE2's dual functions (viral receptor and physiological regulator) require careful consideration to balance antiviral efficacy and potential physiological side effects.

abinScience's ACE2-Related Research Products

Founded in Strasbourg, France, abinScience leverages the region's exceptional research and innovation ecosystem to focus on developing and producing high-quality life science reagents. abinScience offers a range of high-quality research products related to the ACE2 target, including:

       1.   ACE2 Recombinant Proteins: Used for in vitro binding studies, structural research, and drug screening.

       2.   Anti-ACE2 Antibodies: Including monoclonal and polyclonal antibodies, suitable for Western Blot, ELISA, and immunofluorescence experiments to study ACE2 function and expression.

       3.   S Protein-Related Reagents: Such as S protein RBD fragments and full-length proteins, used to investigate S protein-ACE2 interactions.

S Protein-Related Products

References

Xing, L., et al. (2025). Early fusion intermediate of ACE2-using coronavirus spike acting as an antiviral target. *Cell*, 706, 1-18.