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 contains two copies of a sequence motif, the cysteine-histidine box, that is conserved among retroviruses. To identify the functionally relevant positions of a cysteine-histidine box, each amino acid in the proximal copy of the motif was individually substituted by site-directed mutagenesis. Mutations at 5 of 14 positions abolished virus replication and reduced the viral RNA content of mutant particles to between 10 and 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 polyprotein correlated well with the effects of the mutations on particle-associated 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 functionally relevant positions in both motifs led to a significant decline in gag protein export, indicating that the nucleocapsid domain of the gag precursor is also required for efficient assembly or release of the virion.
Dorfman
T
T
Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Luban
J
J
Goff
S P
SP
Haseltine
W A
WA
Göttlinger
H G
HG
D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine; D015683:Gene Products, gag / Q000378:metabolism*; D015750:Genes, gag; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006367:HeLa Cells; D006639:Histidine; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication
D015683:Gene Products, gag; D011498:Protein Precursors; D012367:RNA, Viral; D015678:Viral Structural Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D006639:Histidine; D003545:Cysteine