Cell entry mechanisms of SARS-CoV-2 Shang, Jian; Wan, Yushun; Luo, Chuming ...
Proceedings of the National Academy of Sciences - PNAS,
05/2020, Letnik:
117, Številka:
21
Journal Article
Recenzirano
Odprti dostop
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.
A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-19
. A key to tackling this pandemic is to understand ...the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans
. Here we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
Antibody-dependent enhancement (ADE) of viral entry has been a major concern for epidemiology, vaccine development, and antibody-based drug therapy. However, the molecular mechanism behind ADE is ...still elusive. Coronavirus spike protein mediates viral entry into cells by first binding to a receptor on the host cell surface and then fusing viral and host membranes. In this study, we investigated how a neutralizing monoclonal antibody (MAb), which targets the receptor-binding domain (RBD) of Middle East respiratory syndrome (MERS) coronavirus spike, mediates viral entry using pseudovirus entry and biochemical assays. Our results showed that MAb binds to the virus surface spike, allowing it to undergo conformational changes and become prone to proteolytic activation. Meanwhile, MAb binds to cell surface IgG Fc receptor, guiding viral entry through canonical viral-receptor-dependent pathways. Our data suggest that the antibody/Fc-receptor complex functionally mimics viral receptor in mediating viral entry. Moreover, we characterized MAb dosages in viral-receptor-dependent, Fc-receptor-dependent, and both-receptors-dependent viral entry pathways, delineating guidelines on MAb usages in treating viral infections. Our study reveals a novel molecular mechanism for antibody-enhanced viral entry and can guide future vaccination and antiviral strategies.
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.
The highly contagious and fast-spreading omicron variant of SARS-CoV-2 infects the respiratory tracts efficiently. The receptor-binding domain (RBD) of the omicron spike protein recognizes human ...angiotensin-converting enzyme 2 (ACE2) as its receptor and plays a critical role in the tissue tropism of SARS-CoV-2. Here, we showed that the omicron RBD (strain BA.1) binds to ACE2 more strongly than does the prototypic RBD from the original Wuhan strain. We also measured how individual omicron mutations affect ACE2 binding. We further determined the crystal structure of the omicron RBD (engineered to facilitate crystallization) complexed with ACE2 at 2.6 Å. The structure shows that omicron mutations caused significant structural rearrangements of two mutational hot spots at the RBD/ACE2 interface, elucidating how each omicron mutation affects ACE2 binding. The enhanced ACE2 binding by the omicron RBD may facilitate the omicron variant's infection of the respiratory tracts where ACE2 expression level is low. Our study provides insights into the receptor recognition and tissue tropism of the omicron variant.
Despite the scarcity of the SARS-CoV-2 receptor-human angiotensin-converting enzyme 2 (ACE2)-in the respiratory tract, the omicron variant efficiently infects the respiratory tract, causing rapid and widespread infections of COVID-19. The omicron variant contains extensive mutations in the receptor-binding domain (RBD) of its spike protein that recognizes human ACE2. Here, using a combination of biochemical and X-ray crystallographic approaches, we showed that the omicron RBD binds to ACE2 with enhanced affinity and also elucidated the role of each of the omicron mutations in ACE2 binding. The enhanced ACE2 binding by the omicron RBD may contribute to the omicron variant's new viral tropism in the respiratory tract despite the low level of ACE2 expression in the tissue. These findings help us to understand tissue tropism of the omicron variant and shed light on the molecular evolution of SARS-CoV-2.
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.
Cell entry by coronaviruses involves two principal steps, receptor binding and membrane fusion; the latter requires activation by host proteases, particularly lysosomal proteases. Despite the ...importance of lysosomal proteases in both coronavirus entry and cell metabolism, the correlation between lysosomal proteases and cell tropism of coronaviruses has not been established. Here, we examined the roles of lysosomal proteases in activating coronavirus surface spike proteins for membrane fusion, using the spike proteins from severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) as the model system. To this end, we controlled the contributions from receptor binding and other host proteases, thereby attributing coronavirus entry solely or mainly to the efficiency of lysosomal proteases in activating coronavirus spike-mediated membrane fusion. Our results showed that lysosomal proteases from bat cells support coronavirus spike-mediated pseudovirus entry and cell-cell fusion more effectively than their counterparts from human cells. Moreover, purified lysosomal extracts from bat cells cleave cell surface-expressed coronavirus spikes more efficiently than their counterparts from human cells. Overall, our study suggests that different lysosomal protease activities from different host species and tissue cells are an important determinant of the species and tissue tropism of coronaviruses.
Coronaviruses are capable of colonizing new species, as evidenced by the recent emergence of SARS and MERS coronaviruses; they can also infect multiple tissues in the same species. Lysosomal proteases play critical roles in coronavirus entry by cleaving coronavirus surface spike proteins and activating the fusion of host and viral membranes; they also play critical roles in cell physiology by processing cellular products. How do different lysosomal protease activities from different cells impact coronavirus entry? Here, we controlled the contributions from known factors that function in coronavirus entry so that lysosomal protease activities became the only or the main determinant of coronavirus entry. Using pseudovirus entry, cell-cell fusion, and biochemical assays, we showed that lysosomal proteases from bat cells activate coronavirus spike-mediated membrane fusion more efficiently than their counterparts from human cells. Our study provides the first direct evidence supporting lysosomal proteases as a determinant of the species and tissue tropisms of coronaviruses.
SARS-CoV-2 spike protein plays a key role in mediating viral entry and inducing host immune responses. It can adopt either an open or closed conformation based on the position of its receptor-binding ...domain (RBD). It is yet unclear what causes these conformational changes or how they influence the spike’s functions. Here, we show that Lys417 in the RBD plays dual roles in the spike’s structure: it stabilizes the closed conformation of the trimeric spike by mediating inter-spike–subunit interactions; it also directly interacts with ACE2 receptor. Hence, a K417V mutation has opposing effects on the spike’s function: it opens up the spike for better ACE2 binding while weakening the RBD’s direct binding to ACE2. The net outcomes of this mutation are to allow the spike to bind ACE2 with higher probability and mediate viral entry more efficiently, but become more exposed to neutralizing antibodies. Given that residue 417 has been a viral mutational hotspot, SARS-CoV-2 may have been evolving to strike a balance between infection potency and immune evasion, contributing to its pandemic spread.
The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 ...variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.
Acute diarrhea leads to a substantial disease burden among the elderly worldwide. However, in the context of increasingly aging trend in China, the prevalence of etiological agents among elderly ...diarrheal patients was undetermined. This study aimed to explore the major enteropathogens of acute diarrhea among outpatients older than 65 years in China, and also the epidemiological features of the pathogens. Demographic and clinical data for acute diarrhea among outpatients older than 65 years were collected from 213 participating hospitals from 2009 to 2014. Stool specimens were collected and tested for 13 enteric viruses and bacteria. The proportion of outpatients positive for targeted pathogens was analyzed by residential areas and seasonal patterns. Among the 7,725 patients enrolled, 1,617 (20.9%)were positive for any one of the 13 study pathogens. The predominant pathogen was norovirus (9.0%), followed by diarrheagenic Escherichia coli (DEC) (5.5%), rotavirus (3.9%), non-typhoidal Salmonella (NTS) (2.9%), and Shigella spp. (2.5%). The prevalence of Shigella spp. among rural patients (6.9%) was higher than that among urban patients (1.6%) (p < 0.001), with opposite trend for DEC (3.6% versus 5.9%, p = 0.007). An obvious seasonal pattern was observed for major pathogens, with peak for norovirus in autumn, rotavirus in winter and DEC, NTS, and Shigella spp. in summer. A wide variety of enteropathogens were detected among the elderly with acute diarrhea in China, with norovirus and DEC being the most commonly isolated pathogens. A strong seasonal pattern was observed for major pathogens of acute diarrhea among the elderly.
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