title: Direct and dynamic detection of HIV-1 in living cells.
abstract: In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.
authors.affiliation: Department of Biology II, Ludwig Maximilians University Munich, Planegg-Martinsried, Germany.
authors.forename: Jonas
authors.initials: J
authors.lastname: Helma
authors.forename: Katrin
authors.initials: K
authors.lastname: Schmidthals
authors.forename: Vanda
authors.initials: V
authors.lastname: Lux
authors.forename: Stefan
authors.initials: S
authors.lastname: Nüske
authors.forename: Armin M
authors.initials: AM
authors.lastname: Scholz
authors.forename: Hans-Georg
authors.initials: HG
authors.lastname: Kräusslich
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.forename: Heinrich
authors.initials: H
authors.lastname: Leonhardt
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D015497:HIV-1 / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D021381:Protein Transport; D061905:Single-Domain Antibodies / Q000276:immunology / Q000378:metabolism; D059008:Time-Lapse Imaging; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000276:immunology / Q000378:metabolism
chemical_list: D016655:HIV Core Protein p24; D061905:Single-Domain Antibodies; D054301:gag Gene Products, Human Immunodeficiency Virus