Abstract
The unintegrated viral DNA synthesized during human immunodeficiency virus type 1 infection includes linear and circular forms. Circular forms of viral DNA are surrogate markers for nuclear import of viral DNA during virus replication as well as events surrounding the completion of reverse transcription. Analysis of 2-LTR circles is convenient and the quantity of 2-LTR circle formed is directly proportional to the amount of viral DNA imported into the cell nucleus. In addition, correct synthesis of 2-LTR circles is an outcome of HIV-1 Gag-Pol function. Thus, quantitation and sequence analysis of 2-LTR circles have been very important in studying the structure and function relationship of key viral proteins. In this chapter, we describe the methods of quantitation and analysis of 2-LTR circle junctions isolated from HIV-1 infected cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Goff, S. P. (2001) Intracellular trafficking of retroviral genomes during the early phase of infection: viral exploitation of cellular pathways. J Gene Med 3, 517–552.
Bukrinsky, M. I., Haffar, O. K. (1997) HIV-1 nuclear import: in search of a leader. Front Biosci 2, d578–d587.
Cullen, B. R. (2001) Journey to the center of the cell. Cell 105, 697–700.
Coffin, J. M., Hughes, S. H., Varmus, H. E. (1997) Retroviruses. Cold Harbour laboratory press, Cold Spring Harbour, NY.
Hansen, M. S. T., Carteau, S., Hoffman, C., et al. (1998) Retroviral cDNA integration: mechanism, applications and inhibition. In: Setlow, J.K. (ed.), Genetic Engineering. Principles and Methods, Vol. 20. Plenum Press, New York, NY, pp. 41–62.
Bukrinsky, M. I., Sharova, N., McDonald, T. L., et al. (1993) Association of integrase, matrix, andreverse transcriptase antigens of human immunodeficiency virus type 1 withviral nucleic acids following acute infection. Proc Natl Acad Sci USA 90, 6125–6129.
Li, L., Olvera, J. M., Yoder, K. E., et al. (2001) Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection. EMBO J 20(12), 3272–3281.
Brown, P. O. (1997) Integration. In: Coffin, J.M., Hughes, S.H., Vermus H.E. (eds.), Retroviruses. Cold Spring harbor laboratory press, New York, NY.
Shoemaker, C., Goff, S., Gilboa, E., et al. (1980) Structure of a cloned circular Moloney murine leukemia virus DNA molecule containing an inverted segment: implications for retrovirus integration. Proc Natl Acad Sci USA 77(7), 3932–3936.
Farnet, C. M., Haseltine, W. A. (1991) Circularization of human immunodeficiency virus type 1 DNA in vitro. J Virol 65(12), 6942–6952.
Kilzer, J. M., Stracker, T., Beitzel, B., et al. (2003) Roles of host cell factors in circularization of retroviral DNA. Virology 314(1), 460–467.
Butler, S. L., Hansen, M. S., Bushman, F. D. (2001) A quantitative assay for HIV DNA integration in vivo. Nat Med 7(5), 631–634
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(14), 6537–6546.
Hall, T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucl Acids Symp Ser 41, 95–98.
Brown, T. (1999) Southern Blotting. p 2.9.2–2.9.11. In: Current protocols in molecular biology. Ausubel, F.M (Ed). John Wiley and Sons, Inc.
Zennou, V., Petit, C., Guetard, D., et al. (2000) HIV-1 genome nuclear import is mediated by a central DNA flap. Cell 101(2), 173–185
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
Mandal, D., Prasad, V.R. (2008). Analysis of 2-LTR Circle Junctions of Viral DNA in Infected Cells. 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_6
Download citation
DOI: https://doi.org/10.1007/978-1-59745-170-3_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-859-1
Online ISBN: 978-1-59745-170-3
eBook Packages: Springer Protocols