Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Abstract

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-...

Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins

“…These features may provide a promising landscape on the SARS-CoV-2 S protein for immune recognition. This potential is bolstered by the findings that the convalescent sera from COVID-19 patients contains antibodies against the SARS-CoV-2 S protein (7). A previous study on SARS-CoV has revealed that the oligomannose on the S protein can be recognized by mannose-binding lectin (MBL) and may interfere with viral entry into host cells by inhibition of S protein function (31).…”

SARS-CoV-2: Olfaction, Brain Infection, and the Urgent Need for Clinical Samples Allowing Earlier Virus Detection

“…There is very recent evidence that SARS-CoV-2 enters early and late endosomal compartments in non-neuronal cells; thus, it may possibly be directed to the vesicular axonal pathway in neurons. 1 However, Figure 1. Diagram of human nasal cavity with respiratory and olfactory epithelium areas indicated in blue and yellow, respectively.…”

Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

“…Spike from several coronaviruses can be "pseudotyped" onto safer non-replicative viral particles in place of their endogenous entry protein, thereby making entry of these particles into cells dependent on Spike [29][30][31][32][33][34][35][36]. For SARS-CoV-2, such pseudotyping has recently been reported using HIV-based lentiviral particles [4,27,37], MLV-based retroviral particles [12,38], and VSV [29,[39][40][41]. In the data reported to date, results from such pseudovirus neutralization assays correlate well with measurements made using live SARS-CoV-2 [1,12,27,39].…”