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.
Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, Massachusetts.
T
T
Dorfman
J
J
Luban
S P
SP
Goff
W A
WA
Haseltine
H G
HG
Göttlinger
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