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

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.

Vesicle trafficking and vesicle fusion: mechanisms, biological functions, and their implications for potential disease therapy.

Intracellular vesicle trafficking is the fundamental process to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. These organelles transport cargo from the donor membrane to the target membrane through the cargo containing vesicles. Vesicle trafficking pathway includes vesicle formation from the donor membrane, vesicle transport, and vesicle fusion with the target membrane. Coat protein mediated vesicle formation is a delicate membrane budding process for cargo molecules selection and package into vesicle carriers. Vesicle transport is a dynamic and specific process for the cargo containing vesicles translocation from the donor membrane to the target membrane. This process requires a group of conserved proteins such as Rab GTPases, motor adaptors, and motor proteins to ensure vesicle transport along cytoskeletal track. Soluble N-ethyl-maleimide-sensitive factor (NSF) attachment protein receptors (SNARE)-mediated vesicle fusion is the final process for vesicle unloading the cargo molecules at the target membrane. To ensure vesicle fusion occurring at a defined position and time pattern in eukaryotic cell, multiple fusogenic proteins, such as synaptotagmin (Syt), complexin (Cpx), Munc13, Munc18 and other tethering factors, cooperate together to precisely regulate the process of vesicle fusion. Dysfunctions of the fusogenic proteins in SNARE-mediated vesicle fusion are closely related to many diseases. Recent studies have suggested that stimulated membrane fusion can be manipulated pharmacologically via disruption the interface between the SNARE complex and Ca2+ sensor protein. Here, we summarize recent insights into the molecular mechanisms of vesicle trafficking, and implications for the development of new therapeutics based on the manipulation of vesicle fusion.