CD4 and CCR5 Constitutively Interact at the Plasma Membrane of Living Cells
Gérald GaibeletThierry PlanchenaultSerge MazèresFabrice DumasFernando Arenzana‐SeisdedosAndré LopezBernard LaganeFrançoise Bachelerie
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Abstract:
Human immunodeficiency virus entry into target cells requires sequential interactions of the viral glycoprotein envelope gp120 with CD4 and chemokine receptors CCR5 or CXCR4. CD4 interaction with the chemokine receptor is suggested to play a critical role in this process but to what extent such a mechanism takes place at the surface of target cells remains elusive. To address this issue, we used a confocal microspectrofluorimetric approach to monitor fluorescence resonance energy transfer at the cell plasma membrane between enhanced blue and green fluorescent proteins fused to CD4 and CCR5 receptors. We developed an efficient fluorescence resonance energy transfer analysis from experiments carried out on individual cells, revealing that receptors constitutively interact at the plasma membrane. Binding of R5-tropic HIV gp120 stabilizes these associations thus highlighting that ternary complexes between CD4, gp120, and CCR5 occur before the fusion process starts. Furthermore, the ability of CD4 truncated mutants and CCR5 ligands to prevent association of CD4 with CCR5 reveals that this interaction notably engages extracellular parts of receptors. Finally, we provide evidence that this interaction takes place outside raft domains of the plasma membrane.Keywords:
Lipid raft
Chemokine receptor CCR5
Ectodomain
Herpesvirus glycoprotein B
Fusion mechanism
Viral structural protein
Cell fusion
Viral protein
Viral membrane
Conformational change
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Membrane fusion is an essential step for the entry of enveloped viruses, such as human immunodeficiency virus and influenza virus, into the host cell, often triggered by the binding of membrane proteins on the viral envelope to host cell membrane. Recently, external stimuli was shown to trigger membrane fusion in an artificial system. Direct observation of artificial membrane fusion using a giant unilamellar vesicle (GUV), which is similar in size to a cell, is useful as a biological model system. However, there are no model systems for studying membrane fusion of enveloped viruses with host cells. Here, we report a supramolecular model system for viral entry into a GUV or cell through membrane fusion. The system was constructed by complexing a cationic lipid bilayer on an anionic artificial viral capsid, self-assembled from viral β-annulus peptides. We demonstrate that the cationic enveloped artificial viral capsid electrostatically interacts with the anionic GUV or cell, and the capsid enters the GUV or cell through membrane fusion. The model system established in this study will be important for analyzing membrane fusion during infection of a natural virus.
Fusion mechanism
Viral membrane
Cell fusion
Cell membrane
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Viral protein
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During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing.
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Cell fusion
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In general, enveloped viruses are highly dependent on their lipid envelope for entry into host cells. Here, we demonstrated that during the course of virus maturation, a significant proportion of human herpesvirus 6 (HHV-6) envelope proteins were selectively concentrated in the detergent-resistant glycosphingolipid- and cholesterol-rich membranes (rafts) in HHV-6-infected cells. In addition, the ganglioside GM1, which is known to partition preferentially into lipid rafts, was detected in purified virions, along with viral envelope glycoproteins, gH, gL, gB, gQ1, gQ2 and gO indicating that at least one raft component was included in the viral particle during the assembly process.
Lipid raft
Ganglioside
Raft
Glycosphingolipid
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Viral membrane
Cell fusion
Viral protein
Viral structural protein
Herpesvirus glycoprotein B
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▪ Abstract Viral envelope glycoproteins promote viral infection by mediating the fusion of the viral membrane with the host-cell membrane. Structural and biochemical studies of two viral glycoproteins, influenza hemagglutinin and HIV-1 envelope protein, have led to a common model for viral entry. The fusion mechanism involves a transient conformational species that can be targeted by therapeutic strategies. This mechanism of infectivity is likely utilized by a wide variety of enveloped viruses for which similar therapeutic interventions should be possible.
Herpesvirus glycoprotein B
Fusion mechanism
Infectivity
Viral structural protein
Viral membrane
Viral protein
Cell fusion
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Enveloped animal viruses infect host cells by fusion of viral and target membranes. This crucial fusion event occurs either with the plasma membrane of the host cells at the physiological pH or with the endosomal membranes at low pH and is triggered by specific glycoproteins in the virus envelope. Both lipids and proteins play critical and co-operative roles in the fusion process. Interactions of viral proteins with their receptors direct which membranes fuse and viral fusion proteins then drive the process. These fusion proteins operate on lipid assemblies, whose physical and mechanical properties are equally important to the proper functioning of the process. Lipids contribute to the viral fusion process by virtue of their distinct chemical structure, composition and/or their preferred partitioning into specific microdomains in the plasma membrane called 'rafts'. An involvement of lipid rafts in viral entry and membrane fusion has been examined recently. However, the mechanism(s) by which lipids as dynamic raft components control viral envelope-glycoprotein-triggered fusion is not clear. This paper will review literature findings on the contribution of the two raft-associated lipids, cholesterol and sphingolipids in viral entry.
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Fusion mechanism
Sphingolipid
Viral membrane
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HIV-1 (human immunodeficiency virus type 1) infection begins with the attachment of the virion to a host cell by its envelope glycoprotein (Env), which subsequently induces fusion of viral and cell membranes to allow viral entry. Upon binding to primary receptor CD4 and coreceptor (e.g., chemokine receptor CCR5 or CXCR4), Env undergoes large conformational changes and unleashes its fusogenic potential to drive the membrane fusion. The structural biology of HIV-1 Env and its complexes with the cellular receptors not only has advanced our knowledge of the molecular mechanism of how HIV-1 enters the host cells but also provided a structural basis for the rational design of fusion inhibitors as potential antiviral therapeutics. In this review, we summarize our latest understanding of the HIV-1 membrane fusion process and discuss related therapeutic strategies to block viral entry.
Chemokine receptor CCR5
Rational design
Cell fusion
CCR5 receptor antagonist
Enfuvirtide
Entry inhibitor
Herpesvirus glycoprotein B
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