Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion

Shuai Xia; Meiqin Liu; Chao Wang; Wei Xu; Qiaoshuai Lan; Siliang Feng; Feifei Qi; Linlin Bao; Lanying Du; Shuwen Liu; Chuan Qin; Fei Sun; Zhengli Shi; Yun Zhu; Shibo Jiang; Lu Lu

In April of 2018, the World Health Organization (WHO) established a priority 56 list of pathogens, including Middle East respiratory syndrome (MERS), severe acute 57 respiratory syndrome (SARS) and Disease X, a disease with an epidemic or pandemic 58 potential caused by an unknown pathogen 1,2 (Fig.1a) . 59 In late December 2019, an outbreak of pneumonia with an unknown etiology in 60 Wuhan, China was considered as the first Disease X following the announcement by 61 WHO. Shortly thereafter, a novel coronavirus, 2019-nCoV, as denoted by WHO 3 , 62 was identified as the pathogen causing the coronavirus disease COVID-19 4,5 . 63 2019-nCoV with 79.5% and 96% sequence identity to SARS-CoV and a bat 64 coronavirus, SL-CoV-RaTG13, respectively 6 , was renamed SARS-CoV-2 by the which mediates membrane fusion, has 89.8% sequence identity and 96.9% sequence 83 similarity to those of SARS-CoV, and both of them utilize human 84 angiotensin-converting enzyme 2 (hACE2) as the receptor to infect human cells 6 . 85 Most importantly, the ACE2-binding affinity of the receptor-binding domain (RBD) in 86 S1 subunit of S protein of SARS-CoV-2 is 10-to 20-fold higher than that of 87 SARS-CoV 12 , which may contribute to the higher infectivity and transmissibility of 88 SARS-CoV-2 compared to SARS-CoV. However, it is unclear whether SARS-CoV-2 89 can mediate membrane fusion in a manner that exceeds the capacity of SARS-CoV. 90 After binding of RBD in S1 subunit of S protein on the virion to the ACE2 91 receptor on the target cell, the heptad repeat 1 (HR1) and 2 (HR2) domains in its S2 92 subunit of S protein interact with each other to form a six-helix bundle (6-HB) fusion 93 core, bringing viral and cellular membranes into close proximity for fusion and 94 infection 13 . Therefore, the 6-HB fusion core structure of SARS-CoV-2 and 95 SARS-CoV S proteins should also be compared in order to investigate the structural 96 basis for membrane fusion mediated by their S proteins and thus set the stage for the 97 rational design of coronavirus fusion inhibitors. 98 In our previous studies, we designed a pan-coronavirus fusion inhibitor, EK1, 99 targeting the HR1 domains of HCoV S proteins, which proved to be effective in In this study, we have shown that SARS-CoV-2 exhibits much higher capacity of 108 membrane fusion than SARS-CoV, suggesting that the fusion machinery of SARS-CoV-2 infection, we found a typical syncytium phenomenon naturally formed 139 by infected cells, which is rarely reported in SARS-CoV infection (Fig. 1c) . To 140 further explore the special characteristic of SARS-CoV-2 infection, we cloned the S 141 gene into PAAV-IRES-GFP vector and established the S-mediated cell-cell fusion 142 system, using 293T cells that express SARS-CoV-2 S protein and EGFP 143 (293T/SARS-CoV-2/EGFP) as the effector cells, and ACE2/293T cells expressing 144 human ACE2 receptor as the target cells ( Fig. 1d and Fig. S1a ). After effector cells 145 and target cells were cocultured at 37 °C for 2 h, the fused cells showed at least 2-fold 146 larger size than normal cells and multiple nuclei, and these cells were observed in the 147 SARS-CoV-2 group, but not the SARS-CoV group. After coincubation for 24 h, 148 hundreds of target cells fused together as one big syncytium, containing multiple 149 nuclei (Fig. 1d) . Another 24h later, the syncytium grew bigger and could be easily Previously, we identified that the 6-HB formed by HR1 and HR2 domains of the 166 author/funder. A