Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
McDonald, D., Vodicka, M. A., Lucero, G., et al. (2002) Visualization of the intracellular behavior of HIV in living cells. J Cell Biol 159, 441–452.
Müller, B., Daecke, J., Fackler, O. T., et al. (2004) Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative. J Virol 78, 10803–10813.
Engelman, A., Englund, G., Orenstein, J. M., et al. (1995) Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication. J Virol 69,2729–2736.
Griffin, B. A., Adams, S. R., Tsien, R. Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272.
Arhel, N. J., Genovesio, G., Kim, K. A., et al. (2006) Quantitative 4D tracking of cytoplasmic and nuclear HIV-1 complexes. Nat Meth 3, 817–824.
Rudner, L., Nydegger, S., Coren, L. V., et al. (2005) Dynamic fluorescent imaging of human immunodeficiency virus type 1 gag in live cells by biarsenical labeling. J Virol 79, 4055–4065.
Yee, J. K., Miyanohara, A., LaPorte, P., et al. (1994) A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc Natl Acad Sci U S A 91,9564–9568.
Charneau, P., Mirambeau, G., Roux, P., et al. (1994) HIV-1 reverse transcription. A termination step at the center of the genome. J Mol Biol 241, 651–662.
Adams, S. R., Campbell, R. E., Gross, L. A., et al. (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications. J Amer Chem Soc 124,6063–6076.
Martin, B. R., Giepmans, B. N., Adams, S. R., et al. (2005) Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat Biotechnol 23, 1308–1314.
Arhel, N. J., Munier, S., Souque, P., et al. (2006) Nuclear import defect of DNA Flap mutant HIV-1 viruses is not dependent on the viral strain or target cell type. J Virol 80, 10262–10269.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Arhel, N.J., Charneau, P. (2008). Bisarsenical Labeling of HIV-1 for Real-Time Fluorescence Microscopy. In: Prasad, V.R., Kalpana, G.V. (eds) HIV Protocols. Methods In Molecular Biology™, vol 485. Humana Press. https://doi.org/10.1007/978-1-59745-170-3_11
Download citation
DOI: https://doi.org/10.1007/978-1-59745-170-3_11
Publisher Name: Humana Press
Print ISBN: 978-1-58829-859-1
Online ISBN: 978-1-59745-170-3
eBook Packages: Springer Protocols