Abstract
Virus assembly constitutes a key phase of the HIV-1 replication cycle. The assembly process is initiated by the synthesis of the Gag precursor protein, Pr55Gag, in the cytosol of the infected cell. After its synthesis, Pr55Gag is rapidly transported in most cell types to the plasma membrane (PM) where it associates with the inner leaflet of the lipid bilayer. Gag-Gag interactions lead to the assembly of an electron-dense patch of Gag proteins at the membrane. The viral envelope (Env) glycoproteins associate with Gag during the assembly process. The highly multimerized Gag complex begins to bud outwardly from the PM and eventually pinches off from the cell surface. Concomitant with release, the viral protease cleaves Pr55Gag to the mature Gag proteins matrix, capsid, nucleocapsid and p6, leading to core condensation. The mature infectious virus particle is now able to initiate a new round of infection in a fresh target cell. Techniques have been developed in many laboratories to study each of the distinct phases of the HIV-1 assembly and release pathway. A number of these techniques are described in detail in this chapter.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Freed, E. O. (1998) HIV-1 gag proteins: diverse functions in the virus life cycle. Virology 251, 1–15.
Jouvenet, N., Neil, S. J., Bess, C., et al. (2006) Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly. PLoS Biol 4, e435.
Ono, A., Freed, E. O. (2004) Cell-type-dependent targeting of human immunodeficiency virus type 1 assembly to the plasma membrane and the multivesicular body. J Virol 78, 1552–1563.
Pelchen-Matthews, A., Raposo, G., Marsh, M. (2004) Endosomes, exosomes and Trojan viruses. Trends Microbiol 12, 310–316.
Ono, A., Freed, E. O. (2005) Role of lipid rafts in virus replication. Adv Virus Res 64, 311–358.
Ono, A., Ablan, S. D., Lockett, S. J., et al. (2004) Phosphatidylinositol (4,5) bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proc Natl Acad Sci USA 101, 14889–14894.
Shkriabai, N., Datta, S. A., Zhao, Z., et al. (2006) Interactions of HIV-1 Gag with assembly cofactors. Biochemistry 45, 4077–4083.
Saad, J. S., Miller, J., Tai, J., et al. (2006) Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proc Natl Acad Sci USA 103,11364–11369.
Freed, E. O. (2006) HIV-1 Gag: flipped out for PI(4,5)P(2). Proc Natl Acad Sci USA 103, 11101–11102.
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.
Freed, E. O., Martin, M. A. (1995) Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol 69, 1984–1989.
Huang, M., Orenstein, J. M., Martin, M. A., et al. (1995) p6Gag is required for particle production from full-length human immunodeficiency virus type 1 molecular clones expressing protease. J Virol 69,6810–6818.
Freed, E. O., Orenstein, J. M., Buckler- White, A. J., et al. (1994) Single amino acid changes in the human immunodeficiency virus type 1 matrix protein block virus particle production. J Virol 68, 5311–5320.
von Schwedler, U. K., Stray, K. M., Garrus, J. E., et al. (2003) Functional surfaces of the human immunodeficiency virus type 1 capsid protein. J Virol 77, 5439–5450.
Ono, A., Waheed, A. A., Joshi, A., et al. (2005) Association of human immunodeficiency virus type 1 gag with membrane does not require highly basic sequences in the nucleocapsid: use of a novel Gag multimerization assay. J Virol 79, 14131–14140.
Joshi, A., Nagashima, K., Freed, E. O. (2006) Mutation of dileucine-like motifs in the human immunodeficiency virus type 1 capsid disrupts virus assembly, gag-gag interactions, gag-membrane binding, and virion maturation. J Virol 80, 7939–7951.
Cimarelli, A., Sandin, S., Hoglund, S., et al. (2000) Basic residues in human immunodeficiency virus type 1 nucleocapsid promote virion assembly via interaction with RNA. J Virol 74, 3046–3057.
Ono, A., Orenstein, J. M., Freed, E. O. (2000) Role of the Gag matrix domain in targeting human immunodeficiency virus type 1 assembly. J Virol 74, 2855–2866.
Ono, A., Freed, E. O. (1999) Binding of human immunodeficiency virus type 1 Gag to membrane: role of the matrix amino terminus. J Virol 73, 4136–4144.
Ono, A., Freed, E. O. (2001) Plasma membrane rafts play a critical role in HIV-1 assembly and release. Proc Natl Acad Sci USA 98, 13925–13930.
Yee, J. K., Friedmann, T., Burns, J. C. (1994) Generation of high-titer pseudotyped retroviral vectors with very broad host range. Methods Cell Biol 43, 99–112.
Brown, D. A. (2006) Lipid rafts, detergent-resistant membranes, and raft targeting signals. Physiology (Bethesda) 21, 430–439.
Cimarelli, A., Luban, J. (2000) Human immunodeficiency virus type 1 virion density is not determined by nucleocapsid basic residues. J Virol 74, 6734–6740.
Facke, M., Janetzko, A., Shoeman, R. L., et al. (1993) A large deletion in the matrix domain of the human immunodeficiency virus gag gene redirects virus particle assembly from the plasma membrane to the endoplasmic reticulum. J Virol 67, 4972–4980.
Freed, E. O., Martin, M. A. (2007) HIVs and their replication. in Fields Virology, Fourth Edition, Knipe and Howley, eds. Lippincott Williams & Wilkins.
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
Waheed, A.A., Ono, A., Freed, E.O. (2008). Methods for the Study of HIV-1 Assembly. 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-59745-170-3_12
Publisher Name: Humana Press
Print ISBN: 978-1-58829-859-1
Online ISBN: 978-1-59745-170-3
eBook Packages: Springer Protocols