Unraveling coronavirus cell invasion: The role of glycan receptors in human coronavirus-HKU1 cell entry

Coronaviruses are enveloped RNA viruses distinguished by their crown-like surface spikes. These viruses cause various diseases in humans and animals, including respiratory, gastrointestinal, and neurological disorders. The health risks posed by coronaviruses are substantial, as demonstrated by severe acute respiratory syndrome-coronavirus (SARS-CoV) and SARS-CoV-2. SARS-CoV triggered a global outbreak of SARS in 2003, and SARS-CoV-2 has been responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic since the end of 2019. Furthermore, four human coronaviruses (HCoVs), HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, have been implicated in 15% to 30% cases of the common cold.[1] Given their prevalence and potential to cause various diseases, understanding the mechanisms underlying the invasion into cells of the coronavirus is essential for public health. By binding to specific cell receptors, the spike protein of coronaviruses undergoes conformational changes that promote membrane fusion and entry of the virus into host cells. Although significant progress has been made in elucidating the mechanisms of coronavirus entry, the detailed molecular interactions between viral proteins and host receptors remain incompletely understood. A recent study by Wang et al.[2] reported a critical stage in the HCoV-HKU1 cell invasion process: recognition of the glycan receptor by the spike protein N-terminal domain (NTD). This interaction induces a conformational change crucial for exposing the receptor-binding domain (RBD). Exposure of the RBD to the spike protein of coronaviruses is a critical step that precedes binding to the host receptor, Transmembrane Protease Serine 2 (TMPRSS2). The subsequent binding of the RBD to TMPRSS2 activates the viral fusion machinery, thereby facilitating entry of HCoV-HKU1 into the host cell (Figure 1). This finding has not only deepened understanding of the complex dynamics of viral entry but it also identifies potential targets for the development of antiviral therapies. In the context of concurrent publications in Cell, Wang et al. have provided a novel perspective. While McCallum et al. concentrated on the HCoV-HKU1 spike protein-TMPRSS2 interaction, Wang et al. revealed the synergistic role of glycan receptors in cell invasion, a mechanism not previously explored by McCallum et al.[3] Furthermore, the findings of Wang et al. align with the emphasis by Fernández et al. on activation of TMPRSS2 for HKU1 recognition.[4] By elucidating the synergy between glycan receptors and TMPRSS2, Wang et al. provided a comprehensive view of HCoV-HKU1 cell invasion.Figure 1: Proposed model for human coronavirus (CoV)-HKU1 host cell engagement. The HCoV-HKU1 spike protein interacts with 9-O-acetylsialic acid (9-O-Ac-Sia) via its N-terminal domain (NTD). This interaction triggers an allosteric opening of the adjacent receptor-binding domain (RBD), which facilitates binding to the host cell protease, Transmembrane Protease Serine 2 (TMPRSS2). This figure was created using BioRender.HCoV-HKU1 is a notable human pathogen that causes the common cold, worldwide. The entry mechanism of HCoV-HKU1 into host cells involves a trimeric spike surface glycoprotein that interacts with glycan and proteinaceous receptors.[5,6] Similar to SARS-CoV-2 and Middle East respiratory syndrome (MERS)-CoV, HCoV-HKU1 uses the RBD within the C-terminal domain of the S1 subunit in the spike protein to interact with proteinaceous receptors. In contrast, glycan receptors bind to the NTD of the HCoV-HKU1 spike protein. The glycan receptor, 9-O-acetylsialic acid (9-O-Ac-Sia) interacts with the NTD of HCoV-HKU1, inducing a conformational change that is critical for exposing the RBD.[7] The exposed RBD then interacts with TMPRSS2, leading to activation of the viral fusion machinery, thus facilitating entry into the host cell. Notably, Wang et al. determined the structures of the HCoV-HKU1 spike protein in complex with TMPRSS2 and 9-O-Ac-Sia by cryo-electron microscopy, elucidating the detailed interface between TMPRSS2 and the RBD. Through a meticulous examination of the conserved amino acids, specifically residues 505, 509, 515, 517, and 528 of the HCoV-HKU1A/C spike protein and residues 461, 469, and 470 of the human TMPRSS2 protein at the interface between the RBD of HKU1A and HKU1C and the host receptor TMPRSS2, followed by a series of binding experiments, the functional importance of these pivotal residues was adeptly confirmed. This precise investigation has elucidated their essential role in virus-host receptor interactions, offering profound insights into the molecular underpinnings of entry of viruses into host cells. Wang et al. conducted a comprehensive sequence analysis of the TMPRSS2 interface with the HCoV-HKU1 spike protein across 23 species. They discovered that species, including camel, alpaca, rat, monkey, and pig, whose TMPRSS2 proteins possess key residues, identical to those in the human TMPRSS2 interface, had stronger binding affinity to the RBD. These findings highlight the evolutionary conservation of critical binding sites and their impact on the ability of the virus to interact with various host receptors. These findings offer new insights into potential hosts of HCoV-HKU1. The coronavirus readily undergoes mutations, particularly within the spike protein, potentially altering the infectivity and antigenicity of the virus, subsequently impacting the effectiveness of neutralizing antibodies and vaccines. An array of novel approaches have been used to combat coronaviruses,[8,9] suggesting that a combination of antibodies, vaccines, drugs, or both, simultaneously targeting the TMPRSS2- and sialoglycan-binding sites, could achieve a potent antiviral effect. Building on previous studies, this discovery highlights the crucial role of glycan receptors in the coronavirus infection mechanism. The involvement of glycan receptors in the initial stages of viral attachment and the subsequent facilitation of membrane fusion indicate a complex interplay that is essential for viral success. Understanding the precise mechanisms by which glycan receptors participate in coronavirus cell entry will enhance knowledge of viral pathogenesis and assist in the development of innovative antiviral strategies. The primary glycans involved, include sialic acid, heparan sulfate, chondroitin sulfate proteoglycans, and human histo-blood group antigens. In coronaviruses, heparin sulfate and sialic acid are commonly implicated as glycan receptors during viral invasion of host cells. This specificity is crucial for viral tropism and pathogenicity.[10] Numerous structural studies on glycan binding to spike proteins have elucidated the mechanisms by which these sugar-protein interactions facilitate cell invasion by viruses, thereby enhancing our understanding of glycan-protein dynamics during the entry of viruses into cells. In a pioneering study, Hulswit et al. elucidated the structural basis of 9-O-Ac-Sia interaction with the HCoV-OC43 spike protein.[5] Simultaneously, Park et al. elucidated the structural features of α-2,3- and α-2,6-linked sialic acids that bind to the MERS-CoV spike protein. Their work enriched the understanding of the diverse glycan-binding patterns that may influence viral tropism.[11] Furthermore, Liu et al. delineated the interaction between heparin sulfate and the RBD of the SARS-CoV-2 spike protein, revealing a crucial molecular interaction that the virus exploits to establish an infection in host cells.[12] Previous studies have demonstrated that the spike protein of HCoV-HKU1 undergoes local and long-range conformational changes upon binding to a sialoglycan-based primary receptor at the NTD. This interaction triggers an allosteric shift, leading to a transition of the RBD to an open state (“up”) through interdomain interactions, thereby allowing secondary receptor binding.[7] However, the specific proteinaceous receptors involved, and their detailed structural changes, remain unclear. In 2023, Saunders et al. identified TMPRSS2 as a functional receptor for HCoV-HKU1. Building on these findings, the question arose as to whether the RBD open state induced by sialoglycan influences TMPRSS2 binding to HCoV-HKU1.[6] Wang et al. provided a detailed description of the binding interactions between glycan receptors and the spike protein, highlighting the precise binding structures and interfaces involving the spike protein, 9-O-Ac-Sia, and TMPRSS2.[2] Gao et al. reported similar findings regarding the structural basis for the interaction between HCoV-HKU1 spike RBD and its receptor, TMPRSS2, and proposed an updated model for the dual receptor-based interaction between HCoV-HKU1 and the host cell, providing more detailed insights.[13] Similarly, a seminal 2020 study published in Cell demonstrated that glycan receptors play a crucial role in inducing the exposure of the RBD, thereby assisting in the binding to the proteinaceous receptor. This pivotal finding suggests that heparin sulfate serves as a receptor for SARS-CoV-2. It has been shown that heparin sulfate interacts with the RBD within the S1 subunit of the SARS-CoV-2 spike protein, triggering a conformational change that effectively opens the spike protein structure and allows for subsequent binding to the angiotensin converting enzyme 2 receptor.[12] Since virus-receptor binding is the initial step in infection, targeting glycan receptors such as sialic acid, could potentially prevent entry of viruses into cells. Understanding the synergistic action of glycans and proteinaceous receptors on viral entry has profound implications for antiviral drug development. Targeting the interaction between the coronavirus spike protein and its glycan or proteinaceous receptors could disrupt the initial attachment and prevent the conformational changes necessary for subsequent receptor binding. Additionally, elucidating the structural basis of these interactions could aid in the design of antibodies or small molecules that block the ability of the spike protein to engage with its receptors. Although Zeng et al. provided insights into the role of 9-O-Ac-Sia in HCoV-HKU1 infection, further studies are required to determine whether other coronaviruses use similar invasion mechanisms involving glycan and proteinaceous receptors. Acknowledgments None. Funding This work was supported by grants from the Key Technologies Research and Development Program (2022YFC3400900), Postdoctoral Fellowship Program of CPSF (GZB20230886), China Postdoctoral Science Foundation (2023M743998), National Natural Science Foundation of China (82030046), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019BT02Y198), and Guangdong Science and Technology Department (2020B1212030004). Author Contributions Yan-Lin Yang was responsible for the conceptualization and writing of this research highlight, ensuring that the key findings were accurately summarized and effectively communicated. Zheng-Zhou Lu, Chu Xie and Lan-Yi Zhong provided scientific suggestions for the conceptualization and manuscript revision. Cong Sun contributed through conceptualization, creating figures, and reviewing the manuscript, thereby enhancing the visual appeal and ensuring the accuracy of the presented information. Mu-Sheng Zeng provided expert supervision, reviewing, and editing of this research highlight. All authors have read and approved the final manuscript. Conflicts of Interest None.