Direct and dynamic detection of HIV-1 in living cells

• Published in: PloS one Vol. 7; no. 11; p. e50026
• Main Authors: Helma, Jonas, Schmidthals, Katrin, Lux, Vanda, Nüske, Stefan, Scholz, Armin M, Kräusslich, Hans-Georg, Rothbauer, Ulrich, Leonhardt, Heinrich
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.11.2012; Public Library of Science (PLoS)
• Subjects: Amino Acid Sequence; Antibodies; Antibody Affinity - immunology; Antigens; Biology; Biosensors; Capsid protein; Cell Membrane - metabolism; Cells (biology); Chromatography; Detection equipment; Development and progression; Diagnostic systems; Fluorescence; gag Gene Products, Human Immunodeficiency Virus - immunology; gag Gene Products, Human Immunodeficiency Virus - metabolism; Gag protein; Genetic modification; Genetically modified organisms; Health aspects; HeLa Cells; HIV; HIV Core Protein p24 - chemistry; HIV Core Protein p24 - immunology; HIV Core Protein p24 - metabolism; HIV tests; HIV-1 - immunology; HIV-1 - isolation & purification; HIV-1 - metabolism; Human immunodeficiency virus; Humans; Image analysis; Image processing; Immunoglobulins; Infection; Infectious diseases; Kinetics; Light microscopy; Medicine; Microscopy; Microscopy, Confocal; Molecular Imaging; Molecular Sequence Data; Morphogenesis; Nanobodies; Protein binding; Protein Binding - immunology; Protein Transport; Proteins; Recovery (Medical); Single-Domain Antibodies - immunology; Single-Domain Antibodies - metabolism; Time-Lapse Imaging; Veterinary medicine; Viral proteins; Virions; Virology; Virus Assembly; Viruses; Germany
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0050026

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.