Data Availability StatementAll data are fully available without limitation. pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that this SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S LB42708 protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capability. Also, importantly, the look of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and LB42708 SARS-CoV provides supplied potential arsenals to fight the pandemic and equipment to exploit the fusion system. Research Group (CSG) from the International Committee on Taxonomy of Infections (ICTV). Apr 2020 By 7, a total of just one 1,214,466 verified COVID-19 situations, including 67,767 LB42708 fatalities, have already been reported from 211 countries or locations (www.who.int/emergencies/diseases/novel-coronavirus-2019). The pandemic provides posed significant dangers to global public health and economic and interpersonal stabilities, calling for the urgent development of vaccines and antiviral drugs. CoVs, a large group of enveloped viruses with a single positive-stranded RNA genome, are genetically classified into four genera: (5, 6). The six previously known CoVs that cause human disease include two alphacoronaviruses (human CoV NL63 [HCoV-NL63] and HCoV-229E) and four betacoronaviruses (HCoV-OC43, HCoV-HKU1, SARS-CoV, and Middle East respiratory syndrome CoV [MERS-CoV]). SARS-CoV-2 belongs to the genus and represents the seventh human CoV. Like other CoVs, SARS-CoV-2 uses a glycosylated, homotrimeric class I fusion spike (S) protein to gain entry into host cells (7,C9). The S protein comprises the S1 and S2 subunits and exists in a metastable prefusion conformation. The S1 subunit, which contains a receptor-binding domain name (RBD) capable of functional folding independently, is responsible for virus binding to the cell surface receptor. A recent study suggested that this ACE2-binding affinity of the RBD of SARS-CoV-2 is usually up to 20-fold higher than that of SARS-CoV, which may contribute to its significantly increased infectivity and transmissibility (7). Receptor binding is deemed to trigger large conformational changes in the S complex, which destabilize the prefusion trimer, resulting in shedding of the S1 subunit, and activate the fusogenic activity of the S2 subunit (10,C12). As illustrated in Fig. 1, the sequence structure of S2 contains an N-terminal fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), a transmembrane region (TM), and the cytoplasmic tail (CT). During the fusion process, the FP is usually uncovered and inserts into the target cell membrane, leading S2 in a prehairpin intermediate that bridges the viral and cell membranes; then, three HR1 segments self-assemble a trimeric coiled-coil and three HR2 segments fold into the grooves on the surface of the HR1 inner core, thereby resulting in a six-helical bundle (6-HB) structure that drives the two membranes in close apposition for fusion. SPRY4 Open in a separate windows FIG 1 Schematic diagram of SARS-CoV-2 S protein and its peptide derivatives. (A) Functional domains of the S protein. SP, transmission peptide; NTD, N-terminal domain name; RBD, receptor-binding domain name; SD, subdomain; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain name; CT, cytoplasmic tail. LB42708 The S1/S2 cleavage site (685/686) is usually marked. The HR1 and HR2 sequences and membrane-proximal external sequence (MPES) are outlined. (B) HR2-derived fusion inhibitor peptides. chol, cholesterol. Peptides derived from the HR1 and HR2 sequences of the class I viral fusion proteins have been demonstrated to possess antiviral activity through binding to the prehairpin intermediate, thus blocking the formation of the LB42708 viral 6-HB core (13). These peptides have been discovered to possess activity against rising CoVs also, including SARS-CoV and MERS-CoV (11, 14,C16). In response to.