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

Abstract

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.

Human Immunodeficiency Virus Type 1 Gag Assembly through Assembly Intermediates

“…Consistent with these observations, membranebound Gag complexes have been resolved on Optiprep density gradients (16). However, recent observations of Gag-expressing cells have revealed the occurrence of Gag complexes in the cytoplasm, suggesting Gag multimer formation prior to membrane relocation (17,18).…”

“…However, recent observations of Gag-expressing cells have revealed the occurrence of Gag complexes in the cytoplasm, suggesting Gag multimer formation prior to membrane relocation (17,18). Similarly, data based on the detergent sensitivity of Gag complexes have suggested a detergentresistant complex in the cytosol (19,20), although these cytosolic Gag complexes may possibly be a dead end product (16,21). These apparently conflicting results argue that the morphogenetic pathway of Gag assembly is not as clear cut as is commonly thought, but the data clearly show that some level of Gag multimer, plausibly assembly intermediates, occur during particle assembly.…”

A myristoyl switch regulates membrane binding of HIV-1 Gag

“…Yet a number of questions still remain: (i) When and where does multimerization of nascent Gag polypeptides begin, in the cytosol or at the membrane? Electron microscopy and biochemical studies suggest that newly synthesized Gag is present in small cytosolic complexes and that large-scale multimerization occurs at the membrane (23,24). These data imply that newly synthesized Gag exists in the cytosol in a myr(s) state or perhaps as a small micelle of myr(e) Gag multimers.…”

Properties and functions of the nucleocapsid protein in virus assembly

“…Several cellular proteins were described to interact with the Gag NC domain, which in turn play a role in particle assembly or budding, suggesting that the presence of sequential Gag assembly intermediates. 110,111 Actually, these cellular factors can be eventually incorporated into the nascent budding virions suggesting a dynamic interaction with the assembly complex. 112 The human Staufen 1protein is found in early ribonucleoprotein assembly complexes of HIV-1 as cytoplasmic granules.…”