Interactions of avian myeloblastosis virus nucleocapsid protein with nucleic acids.
The retroviral nucleocapsid protein (NC) associates, histone-like, with genomic RNA within the viral capsid. NC, an essential component of replication competent retroviruses, is also associated with events leading both to virus assembly and to reverse transcription. The nucleic acid binding properties of NC are key to understanding these properties, yet only a minimal biochemical description of NC-nucleic acid interactions is available. We have used the anisotropy of the intrinsic fluorescence of NC from avian myeloblastosis virus to quantify its binding to a variety of nucleic acids. Using salt back-titrations, the intrinsic equilibrium association constant per nucleic acid site, K(obs), was determined for NC binding to single- and double-stranded RNAs and DNAs. In 0.125 M NaCl, 40 mM HEPES at pH 7.0 and 27 degrees C, the log K(obs) ranged from 3.3 to 4.0 (average 3.7) for these nucleic acids. From the salt dependence of K(obs), it was estimated that, on balance, 1 ion was displaced upon formation of each complex; it is likely that cation displacement from nucleic acid is offset by anion binding by protein during complex formation. The logarithm of the mean intrinsic affinity in the absence of polyelectrolyte effects, log KT, was 3.1, corresponding to a delta G of -4.2 kcal/mol. K(obs), KT, and the number of displaced ions were independent of pH between pH 5.6 and 8.9, indicating that NC residues that titrate in this pH range are not contributing to binding. K(obs) and KT increase with temperature, in the range 15 to 47 degrees C. From van't Hoff analysis, entropy was found to be the driving force for formation of the NC-poly(rA) complex, even in the absence of the polyelectrolyte effect. The general nature of NC interactions with nucleic acids is shown by the similarity of the K(obs) values for RNAs and DNAs in both single-stranded and double-stranded structures. This ability of NC to interact with all types of nucleic acids may provide it with the necessary versatility to function like a histone in facilitating the packaging of viral RNA and yet function early in infection, where it has been ascribed a role in facilitating reverse transcription.
Gelfand
C A
CA
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935.
Wang
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Q
Randall
S
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Jentoft
J E
JE
D009189:Avian Myeloblastosis Virus / Q000737:chemistry*; D002213:Capsid / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D009696:Nucleic Acids / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics
D004277:DNA, Single-Stranded; D009696:Nucleic Acids; D011061:Poly A; D012313:RNA; D004247:DNA