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.
Singh
A R
AR
Department of Physiology, University of California at San Francisco, San Francisco, California, 94117, USA.
Hill
R L
RL
Lingappa
J R
JR
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient / Q000379:methods; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015750:Genes, gag; D015488:HIV Antigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus
D015683:Gene Products, gag; D015488:HIV Antigens; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C054858:p17 protein, Human Immunodeficiency Virus Type 1