Mechanisms of SARS-CoV-2 entry into cells

Abstract

The unprecedented public health and economic impact of the COVID-19 pandemic caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been met with an equally unprecedented scientific response. Much of this response has focused, appropriately, on the mechanisms of SARS-CoV-2 entry into host cells, and in particular the binding of the spike (S) protein to its receptor, angiotensin-converting enzyme 2 (ACE2), and subsequent membrane fusion. This Review provides the structural and cellular foundations for understanding the multistep SARS-CoV-2 entry process, including S protein synthesis, S protein structure, conformational transitions necessary for association of the S protein with ACE2, engagement of the receptor-binding domain of the S protein with ACE2, proteolytic activation of the S protein, endocytosis and membrane fusion. We define the roles of furin-like proteases, transmembrane protease, serine 2 (TMPRSS2) and cathepsin L in these processes, and delineate the features of ACE2 orthologues in reservoir animal species and S protein adaptations that facilitate efficient human transmission. We also examine the utility of vaccines, antibodies and other potential therapeutics targeting SARS-CoV-2 entry mechanisms. Finally, we present key outstanding questions associated with this critical process.

Fig. 1. Coronavirus structure and maturation.

Infection by a coronavirus induces in the perinuclear area the formation of new membranous structures of various sizes and shapes, which as a whole are referred to as ‘replication organelles’^–. These structures — observed by electron microscopy in cells infected with mouse hepatitis virus, severe acute respiratory syndrome coronavirus or Middle East respiratory syndrome coronavirus and typically surrounded by double membranes — likely originate from the endoplasmic reticulum (ER) and house viral replication complexes, sequestering them from cellular innate immune molecules. Viral structural proteins and genomic RNA synthesized at the replication site are then translocated through an unknown mechanism to the ER–Golgi intermediate compartment (ERGIC), where virus assembly and budding occur^,. Only four viral proteins — the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins — are incorporated into the virion. While the N protein bound to the viral genomic RNA is packed inside the virion, the structural proteins S, E and M are incorporated in the virion membrane. The S protein, assembled as a trimer, giving the appearance of a crown (corona), mediates major entry steps, including receptor binding and membrane fusion. During biosynthesis and maturation in the infected cell, the S protein is cleaved by furin or furin-like proprotein convertase in the Golgi apparatus into the S1 and S2 subunits, which remain associated^,. The S protein on the virus therefore consists of two non-covalently associated subunits with different functions: in the new target cell, the S1 subunit binds the receptor and the S2 subunit anchors the S protein to the virion membrane and mediates membrane fusion. The E and M proteins contribute to virus assembly and budding through the interactions with other viral proteins^,. Assembled viruses bud into the ERGIC lumen and reach the plasma membrane via the secretory pathway, where they are released into the extracellular space after virus-containing vesicles fuse with the plasma membrane. FP, fusion peptide.

Fig. 2. Structures of the S protein, its subdomains and interaction between the RBD and ACE2.

a | The full-length SARS-CoV-2 spike (S) protein. Selected subdomain structures are shown in ribbon diagrams below the schematic. Three distinct receptor-binding domain (RBD) antigenic sites (AS-1, AS-2 and AS-3) are indicated in the RBD ribbon diagram. b–e | Cryo-electron microscopy structures of detergent-solubilized full-length S trimers from the Wuhan-Hu-1 reference strain in the conformation with three RBDs down (Protein Data Bank (PDB) ID 6XR8; part b), the D614G variant in the conformation with three RBDs down (PDB ID 7KRQ; part c), the D614G variant in the conformation with one RBD up (PDB ID 7KRR; part d) and the postfusion structure of the Wuhan-Hu-1 S2 (PDB ID 6XRA; part e). A dramatic conformational change in heptad repeat 1 (HR1) propels insertion of the fusion peptide into the target membrane. In one protomer, in the foreground, subdomains are coloured according to part a, and the other two protomers, in the background, are shown in grey or light grey. f | The interface between angiotensin-converting enzyme 2 (ACE2; cyan) and bound RBD (red) in the ribbon diagram (PDB ID 6M0J). The receptor-binding motif (RBM) is shown in orange. In the inset, 20 residues of ACE2 and 17 residues from the RBD forming networks of hydrophilic side chain interactions are shown in the stick model. Residues in the RBD that are mutated in the variants of concerns (Table 1) and their interacting residues in ACE2 are highlighted. CH, central helix; CT, cytoplasmic tail; CTD1, carboxy-terminal domain 1; CTD2, carboxy-terminal domain 2; FP, fusion peptide; FPPR, fusion-peptide proximal region; HR2, heptad repeat 2; NTD, amino-terminal domain; S1/S2, furin-cleavage site; S2′, S2′ cleavage site; TM, transmembrane anchor.

Fig. 3. Two distinct SARS-CoV-2 entry pathways.

Two spike (S) protein cleavage events are typically necessary for the coronavirus entry process: one at the junction of the S1 and S2 subunits and the other at the S2′ site, internal to the S2 subunit. In the case of SARS-CoV-2, the polybasic sequence at the S1–S2 boundary is cleaved during virus maturation in an infected cell, but the S2′ site is cleaved at the target cell following angiotensin-converting enzyme 2 (ACE2) binding. Virus binding to ACE2 (step 1) induces conformational changes in the S1 subunit and exposes the S2′ cleavage site in the S2 subunit. Depending on the entry route taken by SARS-CoV-2, the S2′ site is cleaved by different proteases. Left: If the target cell expresses insufficient transmembrane protease, serine 2 (TMPRSS2) or if a virus–ACE2 complex does not encounter TMPRSS2, the virus–ACE2 complex is internalized via clathrin-mediated endocytosis (step 2) into the endolysosomes, where S2′ cleavage is performed by cathepsins, which require an acidic environment for their activity (steps 3 and 4). Right: In the presence of TMPRSS2, S2′ cleavage occurs at the cell surface (step 2). In both entry pathways, cleavage of the S2′ site exposes the fusion peptide (FP) and dissociation of S1 from S2 induces dramatic conformational changes in the S2 subunit, especially in heptad repeat 1, propelling the fusion peptide forward into the target membrane, initiating membrane fusion (step 5 on the left and step 3 on the right). Fusion between viral and cellular membranes forms a fusion pore through which viral RNA is released into the host cell cytoplasm for uncoating and replication (step 6 on the left and step 4 on the right). Several agents disrupt interaction between the S protein and ACE2: ACE2 mimetics, therapeutic monoclonal antibodies targeting the neutralizing epitopes on the S protein and antibodies elicited by vaccination block virus binding to ACE2 and thus inhibit both entry pathways. By contrast, strategies targeting post-receptor-binding steps differ between the two pathways. Being a serine protease inhibitor, camostat mesylate restricts the TMPRSS2-mediated entry pathway. Hydroxychloroquine and chloroquine block endosomal acidification, which is necessary for cathepsin activity, and thus restrict the cathepsin-mediated entry pathway.

Fig. 4. A model for membrane fusion induced by the SARS-CoV-2 S protein.

Structural transition from the prefusion conformation to the postfusion conformation inducing membrane fusion likely proceeds stepwise as follows. The prefusion spike (S) protein trimer fluctuates between the three receptor-binding domain (RBD)-down, closed conformations and one RBD-up, open conformation. RBD binding to angiotensin-converting enzyme 2 (ACE2) enables exposure of the S2′ cleavage site immediately upstream of the adjacent fusion peptide (FP). Cleavage at the S2′ site releases the structural constraints on the FP and initiates a cascade of refolding events in S2, probably accompanied by complete dissociation of S1. Formation of the long central three-stranded coiled coil and folding back of heptad repeat 2 (HR2) leads to the postfusion structure of S2 that brings the two membranes together, facilitating fusion pore formation and viral entry. As shown in Fig. 3, these events can occur either at the plasma membrane or in the endosomal compartment. HR1, heptad repeat 1; TM, transmembrane segment.