Kinetic analysis of human immunodeficiency virus type 1 assembly reveals the presence of sequential intermediates.
The assembly and budding of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1), are mediated by the Gag protein precursor, but the molecular details of these processes remain poorly defined. In this study, we have combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in the lentivirus assembly process. We show that newly synthesized HIV-1 Gag rapidly forms cytoplasmic protein complexes that are resistant to detergent treatment, sensitive to protease digestion, and degraded intracellularly. A subpopulation of newly synthesized Gag binds membranes within 5 to 10 min and over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. These complexes likely represent assembly intermediates because they are not observed with assembly-defective Gag mutants and can be chased into extracellular viruslike particles. At steady state, nearly all of the Gag is present as membrane-bound complexes in various stages of assembly. The identification of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly intermediates should serve as attractive targets for the design of antiviral agents that interfere with the process of particle production.
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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, NY 10021, USA.
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D000818:Animals; D019556:COS Cells; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D003600:Cytosol / Q000378:metabolism; D003902:Detergents; D015683:Gene Products, gag / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D019169:Jurkat Cells; D007700:Kinetics; D017830:Octoxynol; D011092:Polyethylene Glycols; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013997:Time Factors; D014283:Triiodobenzoic Acids; D014357:Trypsin / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology*
D002793:Cholic Acids; D003902:Detergents; D015683:Gene Products, gag; D011498:Protein Precursors; D014283:Triiodobenzoic Acids; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; D014357:Trypsin; C044834:iodixanol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate