Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components

Abstract

The interaction of the retroviral nucleocapsid protein (NC) with nucleic acids forms the basis of its varied roles in the replication cycle, which include binding and condensing the viral RNA within the virion, stimulation of the early steps in reverse transcription, and dissociation from RNA in the replication complex. As part of an investigation of the NC binding site and of the forces that drive its interaction with nucleic acids, the relative affinities of NC from avian myeloblastosis virus were determined for a series of mononucleotides and mononucleotide components using a competitive displacement assay utilizing the extrinsic fluorescent probe bis-ANS [Secnik, J., Wang, Q., Chang, C.-M., & Jentoft, J.E. (1990) Biochemistry 29, 7991-7997]. The estimated binding affinities were unexpectedly similar for nucleotides, nucleosides, and bases (Ka greater than 10(6) M-1). AMP, UMP, GMP, and CMP bound to NC with essentially equal affinity, indicating that NC does not discriminate between bases. This is consistent with its role in coating, condensing, and packaging the RNA within virions. Nucleosides, bases, riboses, and ribose phosphate bind to NC with 1000-fold higher affinity than inorganic phosphate, indicating that the NC binding site includes elements that recognize nucleotide base and ribose components in addition to phosphate ions. However, the binding affinities of components are not additive, i.e., the Kapp values for adenine and deoxyribose are very similar to that for deoxyadenosine, indicating that the interaction between the NC subsite and the base and the sugar components is complex. The stoichiometry of the complex between bis-ANS and NC was established to be NC.(bis-ANS)3.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a Transition State Analog, MgADP-Aluminum Fluoride-Acetate, in Acetate Kinase from Methanosarcina thermophila

“…Two possible explanations for the first phase of fluorescence quenching are: (i) a MgADP-induced environment change around a bound bis-ANS molecule or (ii) displacement of bound bis-ANS by MgADP. In favor of the latter mechanism, it has been reported that bis-ANS binds to hydrophobic pockets and with particularly high affinity to nucleotide binding sites (24,25). Although the results do not distinguish between these two possibilities, the first phase of fluorescence quenching most likely reflects nucleotide binding at the high affinity catalytic site.…”

Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle

“…Similalr levels of protection of the 5' label of ds LTR vere obtained in the presence of levels of NCp7 or NCp7B which corr-esponded to I molecule of NC protein per 13 bp DNA. As the occluded site size of NC protein is approximately 5 -8 bp (26). the DN,As were tfairly well.…”

Temperature Induced Structural Changes ofβ-Crystallin and Sphingomyelin Binding

“…Therefore the phase transition of sphingomyelin is reversible. The temperature dependence of the structure of αcrystallin was studied using a bis-ANS fluorescence probe that has been used to test the surface exposure of hydrophobic sites in proteins (Takashi, Tonomura and Morales, 1977 ;Yoo, Albanesi and Jameson, 1990 ;Secnik, Gelfand and Jentoft, 1992 ;Das and Surewicz, 1995). The anisotropy of bis-ANS increased with an increase of temperature from 22mC to 66mC (Fig.…”