Effect of mutations in Gag on assembly of immature human immunodeficiency virus type 1 capsids in a cell-free system.

Studies of HIV-1 capsid formation in a cell-free system revealed that capsid assembly occurs via an ordered series of assembly intermediates and requires host machinery. Here we use this system to examine 12 mutations in HIV-1 Gag that others studied previously in intact cells. With respect to capsid formation, these mutations generally produced the same phenotype in the cell-free system as in cells, indicating the cell-free system's high degree of fidelity. Analysis of assembly intermediates reveals that a mutation in the distal region of CA (322 LDeltaS) and truncations proximal to the second cys-his box in NC block multimerization of Gag at early stages in the cell-free capsid assembly pathway. In contrast, mutations in the region of amino acids 56-68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations distal to the first cys-his box in NC and mutations in the distal half of MA (88HDeltaG, 85YDeltaG, Delta104-115, and Delta115-129), do not affect formation of immature capsids in the cell-free system. These data provide new information on the role of different domains in Gag during the early events of capsid assembly.