Retroviral nucleocapsid protein specifically recognizes the base and the ribose of mononucleotides and mononucleotide components.
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)
Secnik
J
J
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Gelfand
C A
CA
Jentoft
J E
JE
D000249:Adenosine Monophosphate / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D009189:Avian Myeloblastosis Virus / Q000737:chemistry*; D001665:Binding Sites; D001667:Binding, Competitive; D003568:Cytidine Monophosphate / Q000378:metabolism; D005456:Fluorescent Dyes; D006157:Guanosine Monophosphate / Q000378:metabolism; D008954:Models, Biological; D009711:Nucleotides / Q000378:metabolism*; D012191:Retroviridae Proteins / Q000378:metabolism*; D012266:Ribose / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014542:Uridine Monophosphate / Q000378:metabolism; D014758:Viral Core Proteins / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D009711:Nucleotides; D012191:Retroviridae Proteins; D014758:Viral Core Proteins; D000249:Adenosine Monophosphate; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D012266:Ribose; D006157:Guanosine Monophosphate; D014542:Uridine Monophosphate; D003568:Cytidine Monophosphate