Abstract
The presence of cellular proteins outside and inside retroviruses can indicate the roles they play in viral biology. However, experiments examining retroviruses can be complicated by the contamination of even highly purified virion preparations with nonviral particles (either microvesicles or exosomes). Two useful methods have been developed that can remove contaminating particles from virus stocks to produce highly pure virus preparations. One approach, the subtilisin digestion procedure, enzymatically removes the proteins outside the virions. While this method is well suited for the analysis of the interior proteins in the virions, it removes the extracellular domains of the integral membrane proteins on the virion. To preserve the proteins on the exterior of the virion for biochemical studies, a CD45 immunoaffinity depletion procedure that removes vesicles by capture with antibody-linked microbeads is employed. These methods allow for the isolation of highly purified virion preparations that are suitable for a wide variety of experiments, including the biochemical characterization of cellular proteins both on and in HIV virions, examination of virion/cell interactions, and imaging of virions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ott, D. E. (2002) Potential roles of cellular proteins in HIV-1. Rev Med Virol 12, 359–374.
Bess, J. W., Jr., Gorelick, R. J., Bosche, W. J., et al. (1997) Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 230, 134–144.
Gluschankof, P., Mondor, I., Gelderblom, H. R., et al. (1997) Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations. Virology 230, 125–133.
Trubey, C. M., Chertova, E., Coren, L. V., et al. (2003) Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles. J Virol 77, 12699–12709.
Chertova, E., Chertov, O., Coren, L. V., et al. (2006) Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. J Virol 80, 9039–9052.
Bess, J. W., Jr., Powell, P. J., Issaq, H. J., et al. (1992) Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type 1, and other retroviruses. J Virol 66, 840–847.
Ott, D. E., Coren, L. V., Johnson, D. G., et al. (2000) Actin-binding cellular proteins inside human immunodeficiency virus type 1. Virology 266, 42–51.
Ott, D. E., Coren, L. V., Johnson, D. G., et al. (1995) Analysis and localization of cyclophilin A found in the virions of human immunodeficiency virus type 1 MN strain. AIDS Res Hum Retroviruses 11, 1003–1006.
Ott, D. E., Coren, L. V., Kane, B. P., et al. (1996) Cytoskeletal proteins inside human immunodeficiency virus type 1 virions. J Virol 70, 7734–7743.
Esser, M. T., Graham, D. R., Coren, L.V., et al. (2001) Differential incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation. J Virol 75, 6173–6182.
Ott, D. E., Coren, L. V., Copeland, T. D., et al. (1998) Ubiquitin is covalently attached to the p6Gag proteins of human immunodeficiency virus type-1 and simian immunodeficiency virus and the p12Gag protein of Moloney murine leukemia virus. J Virol 72, 2962–2968.
Ott, D. E., Coren, L. V., Sowder, R. C., II, et al. (2002) Equine infectious anemia virus and the ubiquitin-proteasome system. J Virol 76, 3038–3044.
Acknowledgment
This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 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 organization imply endorsement by the U.S. Government.
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
Ott, D.E. (2008). Purification of HIV-1 Virions by Subtilisin Digestion or CD45 Immunoaffinity Depletion for Biochemical Studies. 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_2
Download citation
DOI: https://doi.org/10.1007/978-1-59745-170-3_2
Publisher Name: Humana Press
Print ISBN: 978-1-58829-859-1
Online ISBN: 978-1-59745-170-3
eBook Packages: Springer Protocols