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.
Highlights
Over the last decades, a large number of HIV (Human Immunodeficiency Virus) detection methodologies have been developed
Gag consists of an N-terminal matrix domain (MA) that mediates membrane attachment, an internal capsid domain (CA) that mediates multimerization of Gag, a nucleocapsid domain (NC) that binds and packages the viral RNA genome and a C-terminal p6 peptide that is involved in virus budding and release
Generation of a CA-specific nanobody In a first step to generate a nanobody reporter for HIV-1 detection in living cells, an alpaca was immunized with purified HIV-1 CA protein and a phagemid library was generated, representing the respective VHH repertoire
Summary
A large number of HIV (Human Immunodeficiency Virus) detection methodologies have been developed. C-terminal insertion of the green fluorescent protein (GFP) as well as internal insertion at the C-terminus of the MA domain allows dynamic visualization of the assembly of virus like particles (VLPs) [6,7,8]. The latter insertion site proved compatible with viral replication and has been used for different tagging strategies, including biarsenical-tetracysteine tagging and SNAP-tagging [9,10]
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