Subtle alterations of the native zinc finger structures have dramatic effects on the nucleic acid chaperone activity of human immunodeficiency virus type 1 nucleocapsid protein.
The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus- and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.
Guo
Jianhui
J
Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Wu
Tiyun
T
Kane
Bradley F
BF
Johnson
Donald G
DG
Henderson
Louis E
LE
Gorelick
Robert J
RJ
Levin
Judith G
JG
D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D018832:Molecular Chaperones; D009154:Mutation; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012357:RNA, Transfer, Lys / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D016335:Zinc Fingers / Q000235:genetics*
D004279:DNA, Viral; D018832:Molecular Chaperones; D019590:Nucleocapsid Proteins; D012357:RNA, Transfer, Lys; D012367:RNA, Viral