Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein.
The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.
Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892.
K
K
Sakaguchi
N
N
Zambrano
E T
ET
Baldwin
B A
BA
Shapiro
J W
JW
Erickson
J G
JG
Omichinski
G M
GM
Clore
A M
AM
Gronenborn
E
E
Appella
D001483:Base Sequence; D001665:Binding Sites; D001973:Bromodeoxyuridine; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D015750:Genes, gag; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D017510:Protein Folding; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014466:Ultraviolet Rays; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D012367:RNA, Viral; D014758:Viral Core Proteins; D001973:Bromodeoxyuridine