Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus

ABSTRACTMultiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections.IMPORTANCEFiloviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

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A screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using a conformation-specific monoclonal antibody

Journal of Virological Methods ◽

10.1016/s0166-0934(99)00041-5 ◽

1999 ◽

Vol 80 (1) ◽

pp. 85-96 ◽

Cited By ~ 80

Author(s):

Shibo Jiang ◽

Kang Lin ◽

Li Zhang ◽

Asim K Debnath

Keyword(s):

Monoclonal Antibody ◽

Core Structure ◽

Specific Monoclonal Antibody ◽

Screening Assay ◽

Antiviral Compounds ◽

Hiv 1

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Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708052114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E7987-E7996 ◽

Cited By ~ 31

Author(s):

Jinwoo Lee ◽

David A. Nyenhuis ◽

Elizabeth A. Nelson ◽

David S. Cafiso ◽

Judith M. White ◽

...

Keyword(s):

Membrane Fusion ◽

Viral Entry ◽

Ebola Virus ◽

Rna Virus ◽

Hemorrhagic Fever ◽

Virus Envelope ◽

Tm Domain ◽

Virus Envelope Protein ◽

Niemann Pick ◽

Fusion Loop

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM–FL interaction in EBOV entry and fusion.

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In Situ Assay of Basic Fibroblast Growth Factor in Cultured Cells with a Specific Monoclonal Antibody.

Cell Structure and Function ◽

10.1247/csf.18.333 ◽

1993 ◽

Vol 18 (5) ◽

pp. 333-343

Author(s):

Masami Minemura ◽

Yoshino Yoshitake ◽

Kouichi Matsuzaki ◽

Akiharu Watanabe ◽

Katsuzo Nishikawa

Keyword(s):

Monoclonal Antibody ◽

Growth Factor ◽

Fibroblast Growth Factor ◽

Basic Fibroblast Growth Factor ◽

Cultured Cells ◽

Fibroblast Growth ◽

Specific Monoclonal Antibody ◽

In Situ Assay

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Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

Proceedings ◽

10.3390/proceedings2020050049 ◽

2020 ◽

Vol 50 (1) ◽

pp. 49

Author(s):

Dibyendu Kumar Das ◽

Uriel Bulow ◽

Natasha D. Durham ◽

Ramesh Govindan ◽

James B. Munro

Keyword(s):

Membrane Fusion ◽

Single Molecule ◽

Conformational Changes ◽

Ebola Virus ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Acidic Ph ◽

Cellular Environment ◽

Niemann Pick

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following the endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. The NPC1 binding to the cleaved GP1 is required for entry, but how this interaction translates to the GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a virus-liposome hemifusion assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding act synergistically to induce conformational changes in GP2 that drive lipid mixing. Acidic pH and Ca2+ shift the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. GP1 cleavage and NPC1 binding enable GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the post-fusion 6-helix bundle. Thus, the GP senses the cellular environment to protect against triggering prior to the arrival of EBOV in a permissive cellular compartment.

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Conformational Dynamics Related to Membrane Fusion Observed in Single Ebola GP Molecules

Proceedings ◽

10.3390/proceedings2020050056 ◽

2020 ◽

Vol 50 (1) ◽

pp. 56

Author(s):

Dibyendu Kumar Das ◽

Uriel Bulow ◽

Natasha D. Durham ◽

Ramesh Govindan ◽

James B. Munro

Keyword(s):

Membrane Fusion ◽

Single Molecule ◽

Conformational Changes ◽

Ebola Virus ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Acidic Ph ◽

Cellular Environment ◽

Niemann Pick

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Sperm proteins SOF1, TMEM95, and SPACA6 are required for sperm−oocyte fusion in mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922650117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11493-11502 ◽

Cited By ~ 10

Author(s):

Taichi Noda ◽

Yonggang Lu ◽

Yoshitaka Fujihara ◽

Seiya Oura ◽

Takayuki Koyano ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Transgenic Mice ◽

Membrane Fusion ◽

Molecular Mechanisms ◽

Cultured Cells ◽

Transmembrane Protein ◽

Sperm Proteins ◽

Sperm Membrane ◽

Oocyte Membrane

Sperm−oocyte membrane fusion is one of the most important events for fertilization. So far, IZUMO1 and Fertilization Influencing Membrane Protein (FIMP) on the sperm membrane and CD9 and JUNO (IZUMO1R/FOLR4) on the oocyte membrane have been identified as fusion-required proteins. However, the molecular mechanisms for sperm−oocyte fusion are still unclear. Here, we show that testis-enriched genes, sperm−oocyte fusion required 1 (Sof1/Llcfc1/1700034O15Rik), transmembrane protein 95 (Tmem95), and sperm acrosome associated 6 (Spaca6), encode sperm proteins required for sperm−oocyte fusion in mice. These knockout (KO) spermatozoa carry IZUMO1 but cannot fuse with the oocyte plasma membrane, leading to male sterility. Transgenic mice which expressed mouseSof1,Tmem95,andSpaca6rescued the sterility ofSof1,Tmem95, andSpaca6KO males, respectively. SOF1 and SPACA6 remain in acrosome-reacted spermatozoa, and SPACA6 translocates to the equatorial segment of these spermatozoa. The coexpression of SOF1, TMEM95, and SPACA6 in IZUMO1-expressing cultured cells did not enhance their ability to adhere to the oocyte membrane or allow them to fuse with oocytes. SOF1, TMEM95, and SPACA6 may function cooperatively with IZUMO1 and/or unknown fusogens in sperm−oocyte fusion.

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Immunocytochemical localization of native chondroitin-sulfate in tissues and cultured cells using specific monoclonal antibody

Cell ◽

10.1016/0092-8674(84)90276-9 ◽

1984 ◽

Vol 38 (3) ◽

pp. 811-822 ◽

Cited By ~ 180

Author(s):

Zafrira Avnur ◽

Benjamin Geiger

Keyword(s):

Monoclonal Antibody ◽

Chondroitin Sulfate ◽

Cultured Cells ◽

Immunocytochemical Localization ◽

Specific Monoclonal Antibody

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A Mucin-Specific Protease Enables Molecular and Functional Analysis of Human Cancer-Associated Mucins

10.26434/chemrxiv.7330676 ◽

2018 ◽

Author(s):

Stacy A. Malaker ◽

Kayvon Pedram ◽

Michael J. Ferracane ◽

Elliot C. Woods ◽

Jessica Kramer ◽

...

Keyword(s):

Mass Spectrometry ◽

Molecular Mechanisms ◽

Cultured Cells ◽

High Resolution Mass Spectrometry ◽

Human Cancer ◽

Glycosylation Sites ◽

Bacterial Protease ◽

Specific Protease ◽

To Come ◽

And Function

<div> <div> <div> <p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular surfaces and are important for human health, especially in the context of carcinomas. However, the molecular mechanisms by which aberrant mucin structures lead to tumor progression and immune evasion have been slow to come to light, in part because methods for selective mucin degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis, and computational peptide docking to demonstrate that a bacterial protease, called StcE, cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure- based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9 receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding receptors. </p> </div> </div> </div>

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