Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2. 1 1234567890():,;C oronaviruses (CoVs) infect human and animals and cause varieties of diseases, including respiratory, enteric, renal, and neurological diseases 1 . They are classified into four genera, alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV 2 . Since beginning of this century, there have already been three zoonotic outbreaks of beta-CoVs. In 2002-2003, severe acute respiratory syndrome coronavirus (SARS-CoV) 3,4 , a lineage B beta-CoV, emerged from bat and palm civet 5,6 , and infected over 8000 people and caused about 800 deaths 7 . In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), a lineage C beta-CoV, was discovered as the causative agent of a severe respiratory syndrome in Saudi Arabia 8 , currently with 2494 confirmed cases and 858 deaths 9 , it remains endemic in Middle East, and dromedary camel is considered as the zoonotic reservoir host of MERS-CoV. At the end of 2019, a novel coronavirus, named SARS-CoV-2, was found in patients with severe pneumonia in Wuhan, China 10-12 . Viruses were isolated from patients and sequenced. Phylogenetical analysis revealed that it is a lineage B beta-CoV and closely related to a SARS-like (SL) CoV, RaTG13, discovered in a cave of Yunnan, China, in 2013 13 . They share about 96% nucleotide sequence identities, suggesting that SARS-CoV-2 might have emerged from a Bat SL-CoV. However, the intermediate host or whether there is an intermediate host remains to be determined.CoV uses its spike glycoprotein (S), a main target for neutralization antibody, to bind its receptor, and mediate membrane fusion and virus entry. Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively. In the structure, N-and C-terminal portions of S1 fold as two independent domains, N-terminal domain (NTD) and C-terminal domain (C-domain) (Fig. 1a). Depending on the virus, either NTD or Cdomain can serve as the receptor-binding domain (RBD). While RBD of mouse hepatitis virus (MHV) is located at the NTD 14 , most of other CoVs, including SARS-CoV and MERS-CoV use C-...

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IMPORTANCEMouse hepatitis virus, a ␤-CoV in group A, uses the galectin-like NTD in its spike protein to bind its receptor protein, while HCoV-OC43, another ␤-CoV in group A, uses the NTD to bind to its sialic-acid containing receptor. In marked contrast, the NTD of the spike glycoprotein of human respiratory ␤-CoV HKU1, which is also in group A, does not bind sugar. In this study, we showed that for the spike protein of HKU1, the purified C domain, downstream of the NTD, could block HKU1 virus infection of human respiratory epithelial cells, and that several monoclonal antibodies that mapped to the C domain neutralized virus infectivity. Thus, the receptor-binding domain of HKU1 spike glycoprotein is located in the C domain. Surprisingly, two ␤-CoVs in group A, mouse hepatitis virus and HKU1, have evolved to use different regions of their spike glycoproteins to recognize their respective receptors. C oronaviruses (CoVs) primarily cause respiratory and enteric diseases in humans, animals, and birds, and some CoVs also cause systemic diseases, including hepatitis or neurological diseases (1). Since the 2002-2003 epidemic of severe acute respiratory syndrome (SARS), intensive surveillance of humans and animals has led to the discovery of numerous other CoVs (2, 3). Phylogenetically, CoVs now are divided into four genera, called the ␣-, ␤-, ␥-, and ␦-CoVs (4). Currently there are six CoVs known to infect humans: two ␣-CoVs, 229E and NL63; two ␤-CoVs in group A, OC43 and HKU1; one ␤-CoV in group B, SARS-CoV; and one ␤-CoV in group C, Middle East respiratory syndrome coronavirus (MERS-CoV), that currently is causing an epidemic with an ϳ30% fatality rate (5-12). While the first four of these human CoVs circulate only in humans and predominately cause mild respiratory diseases, SARS-CoV and MERS-CoV are zoonoses associated with episodically emerging epidemics of severe respiratory infection, including pneumonia, the acute respiratory distress syndrome (ARDS), and death in about 10% to 30% of cases (12,13).The large spikes on the envelope of CoV virions consist of trimers of the ϳ200-kDa spike (S) glycoprotein that bind to hostspecific receptors; mediate virus entry, tissue tropism, and host range; and can affect virus virulence. S protein is the target for CoV neutralizing antibodies and is an essential component of CoV vaccines and vaccine candidates. CoV S proteins are class I viral fusion proteins, like influenza virus hemagglutinin (HA), HIV Env, Ebola virus G, and paramyxovirus F glycoproteins (14). CoV S proteins contain two subunits, called S1 and S2, which are

Human coronavirus (CoV) HKU1 is a pathogen causing acute respiratory illnesses and so far little is known about its biology. HKU1 virus uses its S1 subunit C-terminal domain (CTD) and not the N-terminal domain like other lineage A β-CoVs to bind to its yet unknown human receptor. Here we present the crystal structure of HKU1 CTD at 1.9 Å resolution. The structure consists of three subdomains: core, insertion and subdomain-1 (SD-1). While the structure of the core and SD-1 subdomains of HKU1 are highly similar to those of other β-CoVs, the insertion subdomain adopts a novel fold, which is largely invisible in the cryo-EM structure of the HKU1 S trimer. We identify five residues in the insertion subdomain that are critical for binding of neutralizing antibodies and two residues essential for receptor binding. Our study contributes to a better understanding of entry, immunity and evolution of CoV S proteins.

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