title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.
abstract: The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents. Here we evaluated full length single chain (FLSC) IgG1, a novel IgG-CD4-gp120(BAL) fusion protein with several characteristics that make it an attractive candidate for treatment of HIV-1 infections, including bivalency and a potentially increased serum half-life over FLSC, the parental molecule. FLSC IgG1 binds two domains on CCR5, the N-terminus and the second extracellular loop, lowering the levels of available CCR5 viral attachment sites. Furthermore, FLSC IgG1 synergizes with Maraviroc (MVC), the only licensed CCR5 antagonist. In this study, we used both microscopy and functional assays to address the mechanistic aspects of the interactions of FLSC IgG1 and MVC in the context of CCR5 conformational changes and viral infection. We used a novel stochastic optical reconstruction microscopy (STORM), based on high resolution localization of photoswitchable dyes to visualize direct contacts between FLSC IgG1 and CCR5. We compared viral entry inhibition by FLSC IgG1 with that of other CCR5 blockers and showed FLSC IgG1 to be the most potent. We also showed that lower CCR5 surface densities in HIV-1 infected primary cells result in lower FLSC IgG1 EC50 values. In addition, CCR5 binding by FLSC IgG1, but not CCR5 Ab 2D7, was significantly increased when cells were treated with MVC, suggesting MVC allosterically increases exposure of the FLSC IgG1 binding site. These data have implications for future antiviral therapy development.
authors.affiliation: Institute of Human Virology, University of Maryland, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Olga
authors.initials: O
authors.lastname: Latinovic
authors.affiliation: Institute of Human Virology, University of Maryland, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Kate
authors.initials: K
authors.lastname: Schneider
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Henryk
authors.initials: H
authors.lastname: Szmacinski
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Lakowicz
authors.affiliation: Institute of Human Virology, University of Maryland, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Alonso
authors.initials: A
authors.lastname: Heredia
authors.affiliation: Institute of Human Virology, University of Maryland, University of Maryland School of Medicine, Baltimore, MD, USA; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Redfield
mesh_terms: D015704:CD4 Antigens / Q000235:genetics; D002460:Cell Line; D003510:Cyclohexanes / Q000378:metabolism*; D015483:HIV Antibodies / Q000235:genetics; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics; D000077592:Maraviroc; D018613:Microscopy, Confocal / Q000379:methods; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics; D014230:Triazoles / Q000378:metabolism*; D053586:Virus Internalization / Q000187:drug effects
chemical_list: C584933:CCR5 protein, human; D015704:CD4 Antigens; D003510:Cyclohexanes; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D019713:Receptors, CCR5; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D014230:Triazoles; D000077592:Maraviroc
keywords: CCR5; CCR5 antagonist; Fusion protein; HIV-1; Maraviroc