Lingappa J R, Hill R L, Wong M L, Hegde R S
Department of Physiology, University of California, San Francisco 94143-0444, USA.
J Cell Biol. 1997 Feb 10;136(3):567-81. doi: 10.1083/jcb.136.3.567.
To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of immature HIV capsids de novo in a cell-free system. Capsid authenticity is established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating the fidelity of the cell-free reaction. Assembly is separated into co- and posttranslational phases, and three independent posttranslational requirements are demonstrated: (a) ATP, (b) a detergent-sensitive host factor, and (c) a detergent-insensitive host subcellular fraction that can be depleted and reconstituted. Assembly appears to proceed by way of multiple intermediates whose conversion to completed capsids can be blocked by either ATP depletion or treatment with nondenaturing detergent. Specific subsets of these intermediates accumulate upon expression of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assembly. Furthermore, the accumulation of complexes of similar sizes in cells expressing the corresponding mutants suggests that comparable intermediates may exist in vivo. From these data, we propose a multi-step pathway for the biogenesis of HIV capsids, in which the assembly process can be disrupted at a number of discrete points.
为了解人类免疫缺陷病毒1型（HIV）衣壳形成的机制，我们在无细胞系统中重新构建了未成熟HIV衣壳的从头组装过程。通过多种生化和形态学标准确定了衣壳的真实性。组装过程的已知特征被精确重现，表明无细胞反应的保真度。组装被分为共翻译和翻译后阶段，并证明了三个独立的翻译后要求：（a）ATP，（b）一种对去污剂敏感的宿主因子，以及（c）一种对去污剂不敏感的宿主亚细胞组分，其可以被耗尽并重新构建。组装似乎通过多种中间体进行，其转化为完整衣壳可被ATP耗尽或用非变性去污剂处理所阻断。在无细胞系统中表达各种组装缺陷型Gag突变体时，这些中间体的特定亚集会积累，这表明每个突变体在组装的特定步骤被阻断。此外，在表达相应突变体的细胞中类似大小复合物的积累表明体内可能存在类似的中间体。根据这些数据，我们提出了HIV衣壳生物发生的多步骤途径，其中组装过程可以在多个离散点被破坏。
