A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.
A multistep, ATP-dependent pathway for assembly of human immunodeficiency virus capsids in a cell-free system.
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在无细胞系统中组装人类免疫缺陷病毒衣壳的多步，依赖于ATP的途径。
DOI：
10.1083/jcb.136.3.567
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发表时间：
1997-02-10
期刊：
影响因子：
--
通讯作者：
Hegde RS
中科院分区：
文献类型：
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作者：
Lingappa JR;Hill RL;Wong ML;Hegde RS
为了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组装了未成熟的HIV衣壳。衣壳的真实性是由多种生物化学和形态学标准确定的。组装过程的已知特征被密切再现，表明无细胞反应的保真度。组装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）去污剂敏感的宿主因子，和（c）去污剂不敏感的宿主亚细胞部分，可以耗尽和重建。组装似乎是通过多个中间体进行的，这些中间体转化为完整的衣壳可以通过ATP耗尽或用非变性洗涤剂处理来阻断。这些中间体的特定子集在无细胞系统中表达各种组装缺陷型Gag突变体时积累，表明每个突变体在组装中的特定步骤被阻断。此外，类似大小的复合物在表达相应突变体的细胞中的积累表明，体内可能存在类似的中间体。从这些数据中，我们提出了一个多步骤的途径，为艾滋病毒衣壳的生物合成，其中的组装过程可以在一些离散的点中断。
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.