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

Studies of HIV-I capsid formation in a cell-free system revealed that capsi d assembly occurs via an ordered series of assembly intermediates and requi res host machinery. Here we use this system to examine 12 mutations in HIV- I Gag that others studied previously in intact cells. With respect to capsi d formation, these mutations generally produced the same phenotype in the c ell-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 L DeltaS) and truncations proximal to the seco nd cys-his box in NC block multimerization of Gag at early stages in the ce ll-free capsid assembly pathway. In contrast, mutations in the region of am ino acids 56-68 (located in the proximal portion of MA) inhibit assembly at a later point in the pathway. Other mutations, including truncations dista l to the first cys-his box in NC and mutations in the distal half of MA (88 H DeltaG, 85Y DeltaG, Delta 104-115, and Delta 115-129), do not affect form ation of immature capsids in the cell-free system. These data provide new i nformation on the role of different domains in Gag during the early events of capsid assembly. (C) 2001 Academic Press.