Qibin Geng

A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) is causing the global coronavirus disease 2019 (COVID-19) pandemic. Understanding how SARS-CoV-2 enters human cells is a high priority for deciphering its mystery and curbing its spread. A virus surface spike protein mediates SARS-CoV-2 entry into cells. To fulfill its function, SARS-CoV-2 spike binds to its receptor human ACE2 (hACE2) through its receptor-binding domain (RBD) and is proteolytically activated by human proteases. Here we investigated receptor binding and protease activation of SARS-CoV-2 spike using biochemical and pseudovirus entry assays. Our findings have identified key cell entry mechanisms of SARS-CoV-2. First, SARS-CoV-2 RBD has higher hACE2 binding affinity than SARS-CoV RBD, supporting efficient cell entry. Second, paradoxically, the hACE2 binding affinity of the entire SARS-CoV-2 spike is comparable to or lower than that of SARS-CoV spike, suggesting that SARS-CoV-2 RBD, albeit more potent, is less exposed than SARS-CoV RBD. Third, unlike SARS-CoV, cell entry of SARS-CoV-2 is preactivated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. The high hACE2 binding affinity of the RBD, furin preactivation of the spike, and hidden RBD in the spike potentially allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance. These features may contribute to the wide spread of the virus. Successful intervention strategies must target both the potency of SARS-CoV-2 and its evasiveness.

Antibody-dependent enhancement (ADE) of viral entry has been observed for many viruses. It was shown that antibodies target one serotype of viruses but only subneutralize another, leading to ADE of the latter viruses. Here we identify a novel mechanism for ADE: a neutralizing antibody binds to the surface spike protein of coronaviruses like a viral receptor, triggers a conformational change of the spike, and mediates viral entry into IgG Fc receptor-expressing cells through canonical viral-receptor-dependent pathways. We further evaluated how antibody dosages impacted viral entry into cells expressing viral receptor, Fc receptor, or both receptors. This study reveals complex roles of antibodies in viral entry and can guide future vaccine design and antibody-based drug therapy.

As cell-invading molecular machinery, coronavirus spike proteins pose an evolutionary conundrum due to their high divergence. In this study, we determined the cryo-EM structure of avian infectious bronchitis coronavirus (IBV) spike protein from the γ-genus. The trimeric IBV spike ectodomain contains three receptor-binding S1 heads and a trimeric membrane-fusion S2 stalk. While IBV S2 is structurally similar to those from the other genera, IBV S1 possesses structural features that are unique to different other genera, thereby bridging these diverse spikes into an evolutionary spectrum. Specifically, among different genera, the two domains of S1, the N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), diverge from simpler tertiary structures and quaternary packing to more complex ones, leading to different functions of the spikes in receptor usage and membrane fusion. Based on the above structural and functional comparisons, we propose that the evolutionary spectrum of coronavirus spikes follows the order of α-, δ-, γ-, and β-genus. This study has provided insight into the evolutionary relationships among coronavirus spikes and deepened our understanding of their structural and functional diversity.

Coronavirus spike proteins from different genera are divergent, although they all mediate coronavirus entry into cells by binding to host receptors and fusing viral and cell membranes. Here, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at 3.3-Å resolution. The trimeric protein contains three receptor-binding S1 subunits that tightly pack into a crown-like structure and three membrane fusion S2 subunits that form a stalk. Each S1 subunit contains two domains, an N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD). PdCoV S1-NTD has the same structural fold as alpha- and betacoronavirus S1-NTDs as well as host galectins, and it recognizes sugar as its potential receptor. PdCoV S1-CTD has the same structural fold as alphacoronavirus S1-CTDs, but its structure differs from that of betacoronavirus S1-CTDs. PdCoV S1-CTD binds to an unidentified receptor on host cell surfaces. PdCoV S2 is locked in the prefusion conformation by structural restraint of S1 from a different monomeric subunit. PdCoV spike possesses several structural features that may facilitate immune evasion by the virus, such as its compact structure, concealed receptor-binding sites, and shielded critical epitopes. Overall, this study reveals that deltacoronavirus spikes are structurally and evolutionally more closely related to alphacoronavirus spikes than to betacoronavirus spikes; it also has implications for the receptor recognition, membrane fusion, and immune evasion by deltacoronaviruses as well as coronaviruses in general. IMPORTANCE In this study, we determined the cryo-electron microscopy structure of porcine deltacoronavirus (PdCoV) spike protein at a 3.3-Å resolution. This is the first atomic structure of a spike protein from the deltacoronavirus genus, which is divergent in amino acid sequences from the well-studied alpha- and betacoronavirus spike proteins. Here, we described the overall structure of the PdCoV spike and the detailed structure of each of its structural elements. Moreover, we analyzed the functions of each of the structural elements. Based on the structures and functions of these structural elements, we discussed the evolution of PdCoV spike protein in relation to the spike proteins from other coronavirus genera. This study combines the structure, function, and evolution of PdCoV spike protein and provides many insights into its receptor recognition, membrane fusion, and immune evasion.