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 vi rus type 1 (HIV-1), are mediated by the Gag protein precursor, but the mole cular details of these processes remain poorly defined. In this study, we h ave combined pulse-chase techniques with density gradient centrifugation to identify, isolate, and characterize sequential kinetic intermediates in th e lentivirus assembly process. We show that newly synthesized HIV-1 Gag rap idly forms cytoplasmic protein complexes that are resistant to detergent tr eatment, sensitive to protease digestion, and degraded intracellularly. A s ubpopulation of newly synthesized Gag binds membranes within 5 to 10 min an d over several hours assembles into membrane-bound complexes of increasing size and/or density that can be resolved on Optiprep density gradients. The se complexes likely represent assembly intermediates because they are not o bserved with assembly-defective Gag mutants and can be chased into extracel lular viruslike particles. At steady state, nearly all of the Gag is presen t as membrane-bound complexes in various stages of assembly. The identifica tion of sequential assembly intermediates provides the first demonstration that HIV-1 particle assembly proceeds via an ordered process. Assembly inte rmediates should serve as attractive targets for the design of antiviral ag ents that interfere with the process of particle production.