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© The Rockefeller University Press, 0021-9525/1997//567 $5.00
The Journal of Cell Biology, Volume 136, Number 3, , 1997 567-581

Article

A Multistep, ATP-dependent Pathway for Assembly of Human Immunodeficiency Virus Capsids in a Cell-free System



Jaisri R. Lingappa*,{ddagger}, Rebecca L. Hill*, Mei Lie Wong§, and Ramanujan S. Hegde*

* Department of Physiology, {ddagger} Department of Medicine, and § Department of Biochemistry and HHMI, University of California, San Francisco, California 94143-0444

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.

Abbreviations used in this paper: HBV, Hepatitis B Virus; HIV, human immunodeficiency virus type 1; HSP, high speed pellet; HSS, high speed supernatant; HSPd, detergent-treated high speed pellet; MCoA, myristoyl CoA.

J.R. Lingappa is supported by National Institutes of Health, grant K08AI01292 and R.S. Hegde is supported by Medical Scientist Training Program, grant GM 07618.

Please address all correspondence to Jaisri Lingappa, Department of Physiology and Department of Medicine, University of California, San Francisco, CA 94143-0444. Tel.: (415) 476-4708. Fax.: (415) 476-4929. E-mail: jais{at}itsa.ucsf.edu


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