Abstract
HIV-1 Nucleocapsid protein (NC) is a small basic protein that contains two retroviral zinc fingers. It is a highly effective nucleic acid chaperone that plays a critical role in viral replication acting as a cofactor in reverse transcription as well as other aspects of the viral lifecycle. We have used a variety of biophysical techniques to characterize the high affinity binding of NC to a short deoxyoligonucleotide (d(TG)4). Here we outline in detail the use of fluorescence anisotropy and surface plasmon resonance spectroscopy to study the binding of NC to d(TG)4.
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References
Berkowitz, R., Fisher, J., Goff, S. P. (1996) RNA packaging. Curr. Top. Microbiol. Immunol 214, 177–218
Feng, Y. X., Campbell, S., Harvin, D., et al. (1999) The human immunodeficiency virus type 1 Gag polyprotein has nucleic acid chaperone activity: possible role in dimerization of genomic RNA. J Virol 73, 4251–4256
Cen, S., Huang, Y., Khorchid, A., et al. (1999) The role of Pr55 (gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA. J Virol 73, 4485–4488
Swanstrom, R., Wills, J. W. (1997) Synthesis, assembly, and processing of viral proteins, in Retroviruses. (Coffin JM, Huges SH, Varmus HE. ed.) Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 263–334
Rein, A., Henderson, L. E., Levin, J. G. (1998) Nucleic acid chaperone activity of retroviral nucleocapsid proteins: significance for viral replication. Trends Biochem Sci 23, 297–301
Levin, J. G., Guo, J., Rouzina, I., et al. (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80,217–286
Fisher, R. J., Rein, A., Fivash, M. J., et al. (1998) Sequence-specific binding of human immunodeficiency virus type 1 nucleocapsid protein to short oligonucleotides. J Virol 72, 1902–1909
Fisher, R. J., Fivash, M. J., Stephen, A. G., et al. (2006) Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides. Nucleic Acids Res 34, 472–484
Lakowicz, J. R. (1999) Fluorescence Aniso\-tropy, in Principles of Fluorescence Spectroscopy, 2nd ed., (Lakowicz JR, ed) Kluwer Academic/Plenum Publishers, New York,pp. 291–320
Myszka, D. G. (1999) Improving biosensor analysis. J Mol Rec 12, 279–284
Simpson, J. T., Fisher, R. J. (2005) Measurement of protein-protein interactions using surface plasmon resonance spectroscopy, in Protein-Protein Interactions, 2nd ed. (Golemis EA, Adams PD, ed) Cold Spring Harbor Laboratory Press, Plainview, NY pp. 355–376
Seidel, C. A. M., Schulz, A., Sauer, M. H. M. (1996) Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J Phys Chem 100, 5541–5553
Unruh, J. R., Gokulrangan, G., Lushington, G. H., et al. (2005) Orientational dynamics and dye-DNA interactions in a dye-labeled DNA aptamer. Biophys J 88, 3455–3465
Acknowledgments
We are very grateful to continued support by Matt Fivash on modeling of NC-oligo interactions. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This Research was supported [in part] by the Intramural Research Program and of the NIH, National Cancer Institute, Center for Cancer Research and the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute.
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Stephen, A.G., Fisher, R.J. (2009). Methods for the Analysis of HIV-1 Nucleocapsid Protein Interactions with Oligonucleotides. 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_15
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DOI: https://doi.org/10.1007/978-1-59745-170-3_15
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