Abstract
HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb190) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb190-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb190 and the Rev multimerization domains were evaluated in different assays measuring Nb190-Rev interaction or viral production. Seven residues within Nb190 and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb190-Rev structural interface. This Nb190-Rev interaction model can guide further studies of the Nb190 effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb190 epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Anti-HIV Agents / chemistry
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Anti-HIV Agents / immunology
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Anti-HIV Agents / pharmacology
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Antibody Affinity / immunology
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Cell Line
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Epitope Mapping
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Epitopes / immunology
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HIV-1 / drug effects
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HIV-1 / genetics
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HIV-1 / immunology*
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Humans
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Molecular Docking Simulation
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Molecular Sequence Data
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Mutation
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Protein Binding / immunology
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Protein Conformation
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Protein Transport
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Single-Domain Antibodies / chemistry
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Single-Domain Antibodies / immunology*
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Single-Domain Antibodies / pharmacology
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rev Gene Products, Human Immunodeficiency Virus / chemistry
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rev Gene Products, Human Immunodeficiency Virus / immunology*
Substances
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Anti-HIV Agents
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Epitopes
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Single-Domain Antibodies
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rev Gene Products, Human Immunodeficiency Virus
Grants and funding
Eline Boons was funded by a Ph.D grant of the ‘Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie’ (IWT) (
www.iwt.be). Tom Venken and Arnout Voet thank the research council of the K.U. Leuven for financial support. This work was further supported by the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO) (grant 1.5.104.07 and 1.5.165.10) (
www.fwo.be), the Belgian Government under the framework of the Interuniversity Attraction Poles (I.A.P. P6/19), and the ‘K.U. Leuven Impulsfinanciering’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.