Previously we have described a stepwise, energy-dependent pathway for human immunodeficiency virus type 1 (HIV-1) capsid assembly in a cell-free system. In this pathway, Gag polypeptides utilize the cellular factor HP68 and assemble into immature capsids by way of assembly intermediates that have defined biochemical characteristics. Here we address whether this pathway is universally conserved among primate lentiviruses and can be observed in mammalian cells. We demonstrate that HIV-2 Gag associates with human HP68 in a cell-free system and that Gag proteins of HIV-2, simian immunodeficiency virus SIVmac239, and SIVagm associate with endogenous HP68 in primate cells, as is seen for HIV-1. Analysis of primate cells expressing lentivirus Gag proteins revealed Gag-containing complexes with the same sedimentation values as seen for previously described HIV-1 assembly intermediates in the cell-free system (10S, 80-150S, and 500S). These complexes fit criteria for assembly intermediates as judged by energy sensitivity, pattern of HP68 association, and the failure of specific complexes to be formed by assembly-incompetent Gag mutants. We also demonstrate that virus-like particles released from cells do not appear to contain HP68, suggesting that HP68 is released from Gag upon completion of capsid assembly in cells, as was observed previously in the cell-free system. Together these findings support a model in which all primate lentivirus capsids assemble by a conserved pathway of HP68-containing, energy-dependent assembly intermediates that have specific biochemical features.Human immunodeficiency virus type 1 (HIV-1) particle production can be viewed as a series of distinct biochemical events: first, translation and targeting of newly synthesized Gag polypeptides; next, the posttranslational events of capsid assembly and genome encapsidation; and finally, budding, release, and protease-mediated maturation. With new advances, it is becoming increasingly clear that each of these steps requires complex virus-host interactions (19). This is best exemplified by recent studies showing that budding requires a number of cellular factors, including Tsg101 and the ESCRT proteins (17,41,42,55; reviewed in reference 16).The steps that precede budding are the posttranslational events of immature HIV-1 capsid assembly. The exact mechanism by which Gag assembles posttranslationally in cells remains poorly understood. Capsids of some viruses, such as tobacco mosaic virus (reviewed in reference 32), have been shown to self-assemble, suggesting that the property of capsid protein multimerization is intrinsic to the capsid proteins themselves and is not dependent on other proteins. In the case of retroviruses, a self-assembly model for capsid formation has been proposed based on studies in which purified HIV-1 or Rous sarcoma virus Gag polypeptides and fragments produce spherical structures when incubated with nucleic acid (6, 7, 12, 20-22, 56, 61) and inositol phosphate (5). However, these self-assembly studies were performed with hig...