MAPPING OF FUNCTIONALLY IMPORTANT RESIDUES OF A CYSTEINE-HISTIDINE BOX IN THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NUCLEOCAPSID PROTEIN

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein c ontains two copies of a sequence motif, the cysteine-histidine box, th at is conserved among retroviruses. To identify the functionally relev ant positions of a cysteine-histidine box, each amino acid in the prox imal copy of the motif was individually substituted by site-directed m utagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 a nd 20% of parental levels. Mutations at other positions had either no or only a minor effect on virus replication and virion RNA content. In vitro binding of RNA to bacterially expressed mutant Pr55gag polyprot ein correlated well with the effects of the mutations on particle-asso ciated viral RNA levels. The two different copies of the motif in the HIV-1 nucleocapsid protein are not functionally equivalent, since the conversion of the proximal motif to an exact copy of the distal motif results in a defect in virus replication and a reduction in the viral RNA content of mutant particles. The simultaneous substitution of func tionally relevant positions in both motifs led to a significant declin e in gag protein export, indicating that the nucleocapsid domain of th e gag precursor is also required for efficient assembly or release of the virion.