Kinetic Analysis of Human Immunodeficiency Virus Type 1 Assembly Reveals the Presence of Sequential Intermediates

doi:10.1128/JVI.74.13.5845-5855.2000

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Journal of Virology, July 2000, p. 5845-5855, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Kinetic Analysis of Human Immunodeficiency Virus Type 1 Assembly Reveals the Presence of Sequential Intermediates

Marc Tritel and Marilyn D. Resh^*

Cell Biology Program, Memorial Sloan-Kettering Cancer Center, and Graduate Program in Cell Biology and Genetics, Weill Graduate School of Medical Sciences of Cornell University, New York, New York 10021

Received 14 December 1999/Accepted 29 March 2000

The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag proteinprecursor, but the molecular details of these processes remainpoorly defined. In this study, we have combined pulse-chase techniqueswith density gradient centrifugation to identify, isolate, andcharacterize sequential kinetic intermediates in the lentivirusassembly process. We show that newly synthesized HIV-1 Gag rapidlyforms cytoplasmic protein complexes that are resistant to detergenttreatment, sensitive to protease digestion, and degraded intracellularly.A subpopulation of newly synthesized Gag binds membranes within5 to 10 min and over several hours assembles into membrane-boundcomplexes of increasing size and/or density that can be resolvedon Optiprep density gradients. These complexes likely representassembly intermediates because they are not observed with assembly-defectiveGag mutants and can be chased into extracellular viruslike particles.At steady state, nearly all of the Gag is present as membrane-boundcomplexes in various stages of assembly. The identification ofsequential assembly intermediates provides the first demonstrationthat HIV-1 particle assembly proceeds via an ordered process.Assembly intermediates should serve as attractive targets forthe design of antiviral agents that interfere with the processof particleproduction.

^* Corresponding author. Mailing address: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 143,New York, NY 10021. Phone: (212) 639-2514. Fax: (212) 717-3317.E-mail: m-resh{at}ski.mskcc.org.

Journal of Virology, July 2000, p. 5845-5855, Vol. 74, No. 13
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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