Surfactin Inhibits Membrane Fusion during Invasion of Epithelial Cells by Enveloped Viruses

ABSTRACT Because membrane fusion is a crucial step in the process by which enveloped viruses invade host cells, membrane fusion inhibitors can be effective drugs against enveloped viruses. We found that surfactin from Bacillus subtilis can suppress the proliferation of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in epithelial cells at a relatively low concentration range (15 to 50 μg/ml), without cytotoxicity or viral membrane disruption. Membrane fusion inhibition experiments demonstrate that surfactin treatment significantly reduces the rate at which the virus fuses to the cell membrane. Thermodynamic experiments show that the incorporation of small amounts of surfactin hinders the formation of negative curvature by lamellar-phase lipids, suggesting that surfactin acts a membrane fusion inhibitor. A fluorescent lipopeptide similar to surfactin was synthesized, and its ability to insert into the viral membrane was confirmed by spectroscopy. In vivo experiments have shown that oral administration of surfactin to piglets protects against PEDV infection. In conclusion, our study indicates that surfactin is a membrane fusion inhibitor with activity against enveloped viruses. As the first reported naturally occurring wedge lipid membrane fusion inhibitor, surfactin is likely to be a prototype for the development of a broad range of novel antiviral drugs. IMPORTANCE Membrane fusion inhibitors are a rapidly emerging class of antiviral drugs that inhibit the infection process of enveloped viruses. They can be classified, on the basis of the viral components targeted, as fusion protein targeting or membrane lipid targeting. Lipid-targeting membrane fusion inhibitors have a broader antiviral spectrum and are less likely to select for drug-resistant mutations. Here we show that surfactin is a membrane fusion inhibitor and has a strong antiviral effect. The insertion of surfactin into the viral envelope lipids reduces the probability of viral fusion. We also demonstrate that oral administration of surfactin protects piglets from PEDV infection. Surfactin is the first naturally occurring wedge lipid membrane fusion inhibitor that has been identified and may be effective against many viruses beyond the scope of this study. Understanding its mechanism of action provides a foundation for the development of novel antiviral agents.

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Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

Journal of Virology ◽

10.1128/jvi.00384-20 ◽

2020 ◽

Vol 94 (15) ◽

Cited By ~ 1

Author(s):

Danwei Yu ◽

Jing Xue ◽

Huamian Wei ◽

Zhe Cong ◽

Ting Chen ◽

...

Keyword(s):

Membrane Fusion ◽

Therapeutic Efficacy ◽

Rhesus Macaques ◽

Heptad Repeat ◽

Fusion Inhibitor ◽

Resistance Mutations ◽

Fusion Inhibitors ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

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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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Targeting the Fusion Process of SARS-CoV-2 Infection by Small Molecule Inhibitors

mBio ◽

10.1128/mbio.03238-21 ◽

2022 ◽

Author(s):

Seung Bum Park ◽

Parker Irvin ◽

Zongyi Hu ◽

Mohsin Khan ◽

Xin Hu ◽

...

Keyword(s):

Membrane Fusion ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Host Cells ◽

Fusion Process ◽

Enveloped Viruses ◽

Enveloped Virus ◽

Fusion Inhibitors

SARS-CoV-2 is an enveloped virus that requires membrane fusion for entry into host cells. Since the fusion process is relatively conserved among enveloped viruses, we tested our HCV fusion inhibitors, dichlorcyclizine and fluoxazolevir, against SARS-CoV-2.

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HIV fusion: Catch me if you can

Journal of Biological Chemistry ◽

10.1074/jbc.h120.016022 ◽

2020 ◽

Vol 295 (45) ◽

pp. 15196-15197

Author(s):

Solène Denolly ◽

François-Loïc Cosset

Keyword(s):

Cell Membrane ◽

Membrane Fusion ◽

Lipid Membrane ◽

Electron Tomography ◽

Fusion Process ◽

Target Cells ◽

Tirf Microscopy ◽

Enveloped Viruses ◽

Cryo Electron Tomography

The penetration of enveloped viruses into target cells requires the fusion of the lipid envelope of their virions with the host lipid membrane though a stepwise and highly sophisticated process. However, the intermediate steps in this process have seldom been visualized due to their rarity and rapidity. Here, using cryo-electron tomography, TIRF microscopy, and cell membrane–derived vesicles called blebs, Ward et al. visualize intermediates of the HIV-cell membrane fusion process and demonstrate how Serinc proteins prevent full fusion by interfering with this process.

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Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes

Bioscience Reports ◽

10.1007/bf01114682 ◽

1989 ◽

Vol 9 (3) ◽

pp. 273-305 ◽

Cited By ~ 65

Author(s):

Dick Hoekstra ◽

Jan Willem Kok

Keyword(s):

Membrane Fusion ◽

Membrane Glycoproteins ◽

Enveloped Viruses ◽

Viral Membrane ◽

Intracellular Membranes ◽

Virus Fusion ◽

Specific Proteins ◽

Intracellular Organelles

Enveloped viruses infect cells by a mechanism involving membrane fusion. This process is mediated and triggered by specific viral membrane glycoproteins. Evidence is accumulating that fusion of intracellular membranes, as occurs during endocytosis and transport between intracellular organelles, also requires the presence of specific proteins. The relevance of elucidating the mechanisms of virus fusion for a better understanding of fusion of intracellular membranes is discussed.

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A Robust High-throughput Fluorescent Polarization Assay for the Evaluation and Screening of SARS-CoV-2 Fusion Inhibitors

10.1101/2021.06.17.448891 ◽

2021 ◽

Author(s):

Xinjian Yin ◽

Litong Chen ◽

Siwen Yuan ◽

Lan Liu ◽

Zhizeng Gao

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Global Health ◽

Membrane Fusion ◽

High Throughput ◽

High Throughput Screening ◽

Marine Natural Product ◽

Fusion Inhibitor ◽

Fusion Inhibitors ◽

Natural Product Library ◽

Gain Access

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a serious threat to global health. One attractive antiviral target is the membrane fusion mechanism employed by the virus to gain access to the host cell. Here we report a robust protein-based fluorescent polarization assay, that mimicking the formation of the six-helix bundle (6-HB) process during the membrane fusion, for the evaluation and screening of SARS-CoV-2 fusion Inhibitors. The IC50 of known inhibitors, HR2P, EK1, and Salvianolic acid C (Sal C) were measured to be 6 nM, 2.5 nM, and 8.9 uM respectively. In addition, we found Sal A has a slightly lower IC50 (3.9 uM) than Sal C. Interesting, simple caffeic acid can also disrupt the formation of 6-HB with sub-mM concentration. A pilot high throughput screening (HTS) a small marine natural product library validates the assay with a Z factor close to 0.8. We envision the current assay provides a convenient way to screen SARS-CoV-2 fusion inhibitor and assess their binding affinity.

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A viral regulator of glycoprotein complexes contributes to human cytomegalovirus cell tropism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1419875112 ◽

2015 ◽

Vol 112 (14) ◽

pp. 4471-4476 ◽

Cited By ~ 49

Author(s):

Gang Li ◽

Christopher C. Nguyen ◽

Brent J. Ryckman ◽

William J. Britt ◽

Jeremy P. Kamil

Keyword(s):

Endoplasmic Reticulum ◽

Epithelial Cells ◽

Membrane Fusion ◽

Human Cytomegalovirus ◽

Relative Abundance ◽

Protein Complexes ◽

Cell Tropism ◽

Enveloped Viruses ◽

Viral Glycoproteins ◽

Virion Envelope

Viral glycoproteins mediate entry of enveloped viruses into cells and thus play crucial roles in infection. In herpesviruses, a complex of two viral glycoproteins, gH and gL (gH/gL), regulates membrane fusion events and influences virion cell tropism. Human cytomegalovirus (HCMV) gH/gL can be incorporated into two different protein complexes: a glycoprotein O (gO)-containing complex known as gH/gL/gO, and a complex containing UL128, UL130, and UL131 known as gH/gL/UL128-131. Variability in the relative abundance of the complexes in the virion envelope correlates with differences in cell tropism exhibited between strains of HCMV. Nonetheless, the mechanisms underlying such variability have remained unclear. We have identified a viral protein encoded by theUL148ORF (UL148) that influences the ratio of gH/gL/gO to gH/gL/UL128-131 and the cell tropism of HCMV virions. A mutant disrupted forUL148showed defects in gH/gL/gO maturation and enhanced infectivity for epithelial cells. Accordingly, reintroduction ofUL148into an HCMV strain that lacked the gene resulted in decreased levels of gH/gL/UL128-131 on virions and, correspondingly, decreased infectivity for epithelial cells. UL148 localized to the endoplasmic reticulum, but not to the cytoplasmic sites of virion envelopment. Coimmunoprecipitation results indicated that gH, gL, UL130, and UL131 associate with UL148, but that gO and UL128 do not. Taken together, the findings suggest that UL148 modulates HCMV tropism by regulating the composition of alternative gH/gL complexes.

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Trophoblast Cell Membrane Interactions at Implantation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100068540 ◽

1970 ◽

Vol 28 ◽

pp. 310-311

Author(s):

A. C. Enders

Keyword(s):

Epithelial Cells ◽

Cell Membrane ◽

Membrane Fusion ◽

Trophoblast Cell ◽

Membrane Interactions ◽

Uterine Epithelial Cells ◽

Functional Complex ◽

Cytotrophoblast Cells ◽

Complex Relationships ◽

Syncytial Trophoblast

The alteration in membrane relationships seen at implantation include 1) interaction between cytotrophoblast cells to form syncytial trophoblast and addition to the syncytium by subsequent fusion of cytotrophoblast cells, 2) formation of a wide variety of functional complex relationships by trophoblast with uterine epithelial cells in the process of invasion of the endometrium, and 3) in the case of the rabbit, fusion of some uterine epithelial cells with the trophoblast.Formation of syncytium is apparently a membrane fusion phenomenon in which rapid confluence of cytoplasm often results in isolation of residual membrane within masses of syncytial trophoblast. Often the last areas of membrane to disappear are those including a desmosome where the cell membranes are apparently held apart from fusion.

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