Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity

ABSTRACT The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV 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 the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S 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 capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

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Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity

10.1101/2020.03.26.009233 ◽

2020 ◽

Cited By ~ 4

Author(s):

Yuanmei Zhu ◽

Danwei Yu ◽

Hongxia Yan ◽

Huihui Chong ◽

Yuxian He

Keyword(s):

Membrane Fusion ◽

Cell Receptor ◽

Heptad Repeat ◽

Fusion Inhibitor ◽

Fusion Activity ◽

Global Public Health ◽

S Protein ◽

Entry Pathway ◽

Fusion Inhibitors ◽

A Cell

AbstractThe coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious threats to global public health, economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we firstly verified that SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR1) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly poent activities in inibibiting the SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus infection. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the strcuture and activity relationship (SAR) of IPB02 were characterzized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the presented results have provided important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.

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Blockade of SARS-CoV-2 spike protein-mediated cell–cell fusion using COVID-19 convalescent plasma

Scientific Reports ◽

10.1038/s41598-021-84840-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ling Wang ◽

Juan Zhao ◽

Lam N. T. Nguyen ◽

James L. Adkins ◽

Madison Schank ◽

...

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Neutralizing Antibodies ◽

Molecular Mechanisms ◽

Virus Entry ◽

High Capacity ◽

Target Cells ◽

Global Public Health ◽

S Protein ◽

Cell Cell

AbstractThe recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell–cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell–cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

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A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Journal of Virology ◽

10.1128/jvi.00366-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8303-8314 ◽

Cited By ~ 29

Author(s):

Amanda E. Gardner ◽

Rebecca E. Dutch

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Critical Role ◽

Simian Virus ◽

Hendra Virus ◽

Heptad Repeat ◽

F Protein ◽

Conserved Region ◽

Heptad Repeats ◽

Cell Cell

ABSTRACT Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

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Attenuated fusogenicity and pathogenicity of SARS-CoV-2 Omicron variant

10.21203/rs.3.rs-1207670/v1 ◽

2022 ◽

Author(s):

Kei Sato ◽

Rigel Suzuki ◽

Daichi Yamasoba ◽

Izumi Kimura ◽

Lei Wang ◽

...

Keyword(s):

South Africa ◽

Cell Culture ◽

Severe Acute Respiratory Syndrome ◽

Global Health ◽

Cell Fusion ◽

Statistical Modelling ◽

Hamster Model ◽

S Protein ◽

The Uk ◽

Cell Cell

Abstract The emergence of a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, Omicron, is the most urgent concern in the global health in December 2021. Our statistical modelling estimates that Omicron is >3.0-fold and >5.6-fold more transmissible than Delta in South Africa and the UK, respectively. Intriguingly, cell culture experiments show that Omicron is less fusogenic than Delta and ancestral SARS-CoV-2. Although the spike (S) protein of Delta is efficiently cleaved into the two subunits, which facilitates cell-cell fusion, Omicron S is faintly cleaved. Further, in hamster model, Omicron shows decreased lung infectivity and is less pathogenic compared to Delta and ancestral SARS-CoV-2. Our data suggest that the efficacy of SARS-CoV-2 S cleavage and viral fusogenicity are closely associated with viral pathogenicity, and Omicron evolved to exhibit increased transmissibility and attenuated pathogenicity.

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Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

Journal of Virology ◽

10.1128/jvi.80.3.1302-1310.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1302-1310 ◽

Cited By ~ 38

Author(s):

Rene Broer ◽

Bertrand Boson ◽

Willy Spaan ◽

François-Loïc Cosset ◽

Jeroen Corver

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Membrane Fusion ◽

Cell Fusion ◽

Transmembrane Domain ◽

Cytoplasmic Tail ◽

Transmembrane Domains ◽

Spike Protein ◽

Fusion Activity ◽

Wild Type ◽

Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

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Cell-Cell Fusion Induced by the Avian Reovirus Membrane Fusion Protein Is Regulated by Protein Degradation

Journal of Virology ◽

10.1128/jvi.78.11.5996-6004.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 5996-6004 ◽

Cited By ~ 22

Author(s):

Maya Shmulevitz ◽

Jennifer Corcoran ◽

Jayme Salsman ◽

Roy Duncan

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Transmembrane Protein ◽

Syncytium Formation ◽

Replication Cycle ◽

Stable Form ◽

Avian Reovirus ◽

Membrane Fusion Protein ◽

Cell Cell

ABSTRACT The p10 fusion-associated small transmembrane protein of avian reovirus induces extensive syncytium formation in transfected cells. Here we show that p10-induced cell-cell fusion is restricted by rapid degradation of the majority of newly synthesized p10. The small ectodomain of p10 targets the protein for degradation following p10 insertion into an early membrane compartment. Paradoxically, conservative amino acid substitutions in the p10 ectodomain hydrophobic patch that eliminate fusion activity also increase p10 stability. The small amount of p10 that escapes intracellular degradation accumulates at the cell surface in a relatively stable form, where it mediates cell-cell fusion as a late-stage event in the virus replication cycle. The unusual relationship between a nonstructural viral membrane fusion protein and the replication cycle of a nonenveloped virus has apparently contributed to the evolution of a novel mechanism for restricting the extent of virus-induced cell-cell fusion.

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Novel roles of the N1 loop and N4 alpha helical region of the Nipah Virus Fusion glycoprotein in modulating early and late steps of the membrane fusion cascade

Journal of Virology ◽

10.1128/jvi.01707-20 ◽

2021 ◽

Author(s):

J. Lizbeth Reyes Zamora ◽

Victoria Ortega ◽

Gunner P. Johnston ◽

Jenny Li ◽

Hector C. Aguilar

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Receptor Binding ◽

Viral Entry ◽

Nipah Virus ◽

Host Cells ◽

Fusion Pore ◽

Loop Region ◽

Cell Cell

Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae. NiV is deadly to humans, infecting host cells by direct fusion of the viral and host-cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion-pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the HR1 and HR2 regions in the fusion protein pre-fusion conformation bind to form a six-helix bundle in the post-fusion conformation. Here, structural comparisons between the F pre-fusion and post-fusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization, and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scan mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion-pore expansion during syncytia formation, also uncovering a link between F/G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections. IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching ∼75% in humans. Known human outbreaks have concentrated in southeast Asia and Australia. Further, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important process of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.

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Insertion of the Two Cleavage Sites of the Respiratory Syncytial Virus Fusion Protein in Sendai Virus Fusion Protein Leads to Enhanced Cell-Cell Fusion and a Decreased Dependency on the HN Attachment Protein for Activity

Journal of Virology ◽

10.1128/jvi.00078-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5986-5998 ◽

Cited By ~ 22

Author(s):

Joanna Rawling ◽

Blanca García-Barreno ◽

José A. Melero

Keyword(s):

Respiratory Syncytial Virus ◽

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Sendai Virus ◽

Cleavage Sites ◽

Attachment Protein ◽

F Protein ◽

Syncytial Virus ◽

Cell Cell

ABSTRACT Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (FRSV) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (FSeV), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both FRSV cleavage sites in FSeV resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric FSeV mutants containing both FRSV cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

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Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide

Journal of Virology ◽

10.1128/jvi.01026-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 8

Author(s):

Michael N. Ha ◽

Sébastien Delpeut ◽

Ryan S. Noyce ◽

Gary Sisson ◽

Karen M. Black ◽

...

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Cell Lines ◽

Measles Virus ◽

Structural Changes ◽

3D Structure ◽

Syncytium Formation ◽

Heptad Repeat ◽

Fusion Inhibitors ◽

Site Of Action

ABSTRACT The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

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Reptilian Reovirus Utilizes a Small Type III Protein with an External Myristylated Amino Terminus To Mediate Cell-Cell Fusion

Journal of Virology ◽

10.1128/jvi.78.8.4342-4351.2004 ◽

2004 ◽

Vol 78 (8) ◽

pp. 4342-4351 ◽

Cited By ~ 66

Author(s):

Jennifer A. Corcoran ◽

Roy Duncan

Keyword(s):

Fusion Protein ◽

Membrane Fusion ◽

Cell Fusion ◽

Fusion Proteins ◽

Signal Sequence ◽

Topological Analysis ◽

Protein Family ◽

Sequence Comparisons ◽

Type Iii ◽

Cell Cell

ABSTRACT Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins Nexoplasmic/Ccytoplasmic (Nexo/Ccyt), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.

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