Sequence-specific binding of human immunodeficiency virus type 1 nucleocapsid protein to short oligonucleotides.
We have analyzed the binding of recombinant human immunodeficiency virus type 1 nucleocapsid protein (NC) to very short oligonucleotides by using surface plasmon resonance (SPR) technology. Our experiments, which were conducted at a moderate salt concentration (0.15 M NaCl), showed that NC binds more stably to runs of d(G) than to other DNA homopolymers. However, it exhibits far more stable binding with the alternating base sequence d(TG)n than with any homopolymeric oligodeoxyribonucleotide; thus, it shows a strong sequence preference under our experimental conditions. We found that the minimum length of an alternating d(TG) sequence required for stable binding was five nucleotides. Stable binding to the tetranucleotide d(TG)2 was observed only under conditions where two tetranucleotide molecules were held in close spatial proximity. The stable, sequence-specific binding to d(TG)n required that both zinc fingers be present, each in its proper position in the NC protein, and was quite salt resistant, indicating a large hydrophobic contribution to the binding. Limited tests with RNA oligonucleotides indicated that the preferential sequence-specific binding observed with DNA also occurs with RNA. Evidence was also obtained that NC can bind to nucleic acid molecules in at least two distinct modes. The biological significance of the specific binding we have detected is not known; it may reflect the specificity with which the parent Gag polyprotein packages genomic RNA or may relate to the functions of NC after cleavage of the polyprotein, including its role as a nucleic acid chaperone.
Fisher
R J
RJ
Protein Chemistry Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Maryland 21702, USA. fisher@ncifcrf.gov
Rein
A
A
Fivash
M
M
Urbaneja
M A
MA
Casas-Finet
J R
JR
Medaglia
M
M
Henderson
L E
LE
D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000378:metabolism; D005453:Fluorescence; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D012313:RNA