MULTISTEP, ATP-DEPENDENT PATHWAY FOR ASSEMBLY OF HUMAN-IMMUNODEFICIENCY-VIRUS CAPSIDS IN A CELL-FREE SYSTEM

To understand the mechanism by which human immunodeficiency virus type 1 (HIV) capsids are formed, we have reconstituted the assembly of imm ature HIV capsids de novo in a cell-free system. Capsid authenticity i s established by multiple biochemical and morphologic criteria. Known features of the assembly process are closely reproduced, indicating th e fidelity of the cell-free reaction. Assembly is separated into co- a nd posttranslational phases, and three independent posttranslational r equirements 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 b e blocked by either ATP depletion or treatment with nondenaturing dete rgent. Specific subsets of these intermediates accumulate upon express ion of various assembly-defective Gag mutants in the cell-free system, suggesting that each mutant is blocked at a particular step in assemb ly. Furthermore, the accumulation of complexes of similar sizes in cel ls expressing the corresponding mutants suggests that comparable inter mediates may exist in vivo. From these data, we propose a multi-step p athway for the biogenesis of HIV capsids, in which the assembly proces s can be disrupted at a number of discrete points.