Bisarsenical labeling of HIV-1 for real-time fluorescence microscopy.
Imaging studies have benefited from the development of a novel technique for non-destructive labeling of proteins within living cells, based on the use of a reagent called FlAsH-EDT2, a bisarsenical derivative of fluorescein capable of binding with high affinity and specificity to a tetracysteine motif in the protein of interest. This technique has been adapted for the stable, sensitive and specific molecular tagging of HIV-1 IN enabling the tracking of incoming viral particles inside infected living cells. Here we present the experimental steps required for the efficient labeling of HIV-1 IN, namely, molecular insertion of a tetracysteine tag, production of viruses, labeling in vitro of tagged viruses, infection of target cells and visualization of particles by fluorescence microscopy.
Arhel
Nathalie J
NJ
Virology Department, Institut Pasteur, Molecular Virology and Vectorology Group, Paris, France.
Charneau
Pierre
P
D002460:Cell Line; D005452:Fluoresceins / Q000378:metabolism*; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D009942:Organometallic Compounds / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
C113276:4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein; D005452:Fluoresceins; D009942:Organometallic Compounds; D019427:HIV Integrase