Abstract
A discerning feature of the retrovirus lifecycle is the covalent integration of the viral reverse transcript into a chromosome within the infected cell. Integration is required for productive infection and therefore defines the viral integrase protein of human immunodeficiency virus type 1 (HIV-1) as a bona fide target for the development of antiviral drugs in the fight against HIV/AIDS. Integrase works in the context of the viral preintegration complex (PIC), a high molecular weight nucleoprotein complex that supports the integration of its endogenous viral DNA copy made during reverse transcription into an exogenous target DNA in the test tube. PIC analyses are central to understanding the molecular mechanisms of HIV-1 integration as well as investigating the pharmacological properties of integrase inhibitors. This chapter describes techniques for isolating HIV-1 PICs from cells as well as quantifying their level of integration activity in vitro.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Katzman, M. and Katz, R. A. (1999) Substrate recognition by retroviral integrases. Adv Virus Res 52, 371–395.
Pauza, C. (1990) Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination. Virology 179, 886–889.
Miller, M., Farnet, C., Bushman, F. (1997) Human immunodeficiency virus type 1 preintegration complexes: studies of organization and composition. J Virol 71, 5382–5390.
Fujiwara, T., Mizuuchi, K. (1988) Retroviral DNA integration: structure of an integration intermediate. Cell 54, 497–504.
Brown, P. O., Bowerman, B., Varmus, H. E., et al. (1989) Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein. Proc Natl Acad Sci USA 86, 2525–2529.
Lee, Y. M., Coffin, J. M. (1991) Relationship of avian retrovirus DNA synthesis to integration in vitro. Mol Cell Biol 11, 1419–1430.
Yoder, K. E., Bushman, F. D. (2000) Repair of gaps in retroviral DNA integration intermediates. J Virol 74, 11191–11200.
Turlure, F., Devroe, E., Silver, P. A., et al. (2004) Human cell proteins and human immunodeficiency virus DNA integration. Front Biosci 9, 3187–3208.
Brown, P. O., Bowerman, B., Varmus, H. E., et al. (1987) Correct integration of retroviral DNA in vitro. Cell 49, 347–356.
Ellison, V., Abrams, H., Roe, T., Lifson, J., et al. (1990) Human immunodeficiency virus integration in a cell-free system. J Virol 64, 2711–2715.
Farnet, C. M., Haseltine, W. A. (1990) Integration of human immunodeficiency virus type 1 DNA in vitro. Proc Natl Acad Sci USA 87, 4164–4168.
Hansen, M. S., Smith, G. J., Kafri, T., et al. (1999) Integration complexes derived from HIV vectors for rapid assays in vitro. Nat Biotechnol 17, 578–582.
Brooun, A., Richman, D. D., Kornbluth, R. S. (2001) HIV-1 preintegration complexes preferentially integrate into longer target DNA molecules in solution as detected by a sensitive, polymerase chain reaction-based integration assay. J Biol Chem 276, 46946–46952.
Lu, R., Vandegraaff, N., Cherepanov, P., et al. (2005) Lys-34, dispensable for integrase catalysis, is required for preintegration complex function and human immunodeficiency virus type 1 replication. J Virol 79, 12584–12591.
Dismuke, D. J., Aiken, C. (2006) Evidence for a functional link between uncoating of the human immunodeficiency virus type 1 core and nuclear import of the viral preintegration complex. J Virol 80, 3712–3720.
Oh, J., Chang, K. W., Hughes, S. H. (2006) Mutations in the U5 sequences adjacent to the primer binding site do not affect tRNA cleavage by Rous sarcoma virus RNase H but do cause aberrant integrations in vivo. J Virol 80, 451–459.
Chen, H., Wei, S.-Q., Engelman, A. (1999) Multiple integrase functions are required to form the native structure of the human immunodeficiency virus type I intasome. J Biol Chem 274, 17358–17364.
Salahuddin, S. Z., Markham, P. D., Wong-Staal, F., et al. (1983) Restricted expression of human T-cell leukemia–lymphoma virus (HTLV) in transformed human umbilical cord blood lymphocytes. Virology 129, 51–64.
Adachi, A., Gendelman, H. E., Koenig, S., et al. (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol 59, 284–291.
Dorfman, T., Luban, J., Goff, S. P., et al. (1993) Mapping of functionally important residues of a cysteine-histidine box in the human immunodeficiency virus type 1 nucleocapsid protein. J Virol 67, 6159–6169.
Julias, J. G., Ferris, A. L., Boyer, P. L., et al. (2001) Replication of phenotypically mixed human immunodeficiency virus type 1 virions containing catalytically active and catalytically inactive reverse transcriptase. J. Virol. 75, 6537–6546.
Butler, S. L., Hansen, M. S. T., Bushman, F. D. (2001) A quantitative assay for HIV DNA integration in vivo. Nat Med 7, 631–634.
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.
Miller, M. D., Wang, B., Bushman, F. D. (1995) Human immunodeficiency virus type 1 preintegration complexes containing discontinuous plus strands are competent to integrate in vitro. J Virol 69, 3938–3944.
Harrison, G. P., Mayo, M. S., Hunter, E., et al. (1998) Pausing of reverse transcriptase on retroviral RNA templates is influenced by secondary structures both 5′ and 3′ of the catalytic site. Nucleic Acids Res 26, 3433–3442.
Byers, K. B., Engelman, A., Fontes, B. (2004) General guidelines for experimenting with HIV, in Protocols in Immunology Supplement 59 (Coligan, J. E., Bierer, B. E., Margulies, D. H., Shevach, E. M., and Strober, W., eds.), John Wiley & Sons, Hoboken, NJ, pp. 12.1.1–12.1.9.
O’Doherty, U., Swiggard, W. J., Malim, M. H. (2000) Human immunodeficiency virus type 1 spinoculation enhances infection through virus binding. J Virol 74, 10074–10080.
Sambrook, J., Fritsch, E. F., Maniatis, T. (eds.) (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 9.34–9.36.
Wei, S.-Q., Mizuuchi, K., Craigie, R. (1997) A large nucleoprotein assembly at the ends of the viral DNA mediates retroviral DNA integration. EMBO J 16, 7511–7520.
Chen, H., Engelman, A. (2001) Asymmetric processing of human immunodeficiency virus type 1 cDNA in vivo: implications for functional end coupling during the chemical steps of DNA transposition. Mol Cell Biol 21, 6758–6767.
Bao, K. K., Wang, H., Miller, J. K., Erie, D. A., Skalka, A. M., and Wong, I. (2003) Functional oligomeric state of avian sarcoma virus integrase. J Biol Chem 278, 1323–1327.
Faure, A., Calmels, C., Desjobert, C., et al. (2005) HIV-1 integrase crosslinked oligomers are active in vitro. Nucleic Acids Res 33, 977–986.
Guiot, E., Carayon, K., Delelis, O., et al. (2006) Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity. J Biol Chem 281, 22707–22719.
Li, M., Mizuuchi, M., Burke, T. R. J., et al. (2006) Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J 25, 1295–1304.
Vincent, K. A., York-Higgins, D., Quiroga, M., et al. (1990) Host sequences flanking the HIV provirus. Nucleic Acids Res 18, 6045–6047.
Vink, C., Groenink, M., Elgersma, Y., et al. (1990) Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA. J Virol 64, 5626–5627.
Acknowledgments
I thank J.E. Daigle, K. McGee-Estrada, and N.K. Raghavendra for critically reading the manuscript. This work was supported by NIH grants AI39394, AI52014, and AI70042.
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
Engelman, A. (2008). Isolation and Analysis of HIV-1 Preintegration Complexes. 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_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-170-3_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-859-1
Online ISBN: 978-1-59745-170-3
eBook Packages: Springer Protocols