Document 3281 DOCN M94A3281 TI Mutations of basic amino acids of NCp7 of HIV-1 affect RNA binding in vitro. DT 9412 AU Schmalzbauer E; Dannull J; Guehmann S; Moelling K; Max-Planck-Institute for Molecular Genetics, Berlin, FRG. SO Int Conf AIDS. 1994 Aug 7-12;10(1):111 (abstract no. PA0061). Unique Identifier : AIDSLINE ICA10/94369294 AB OBJECTIVES: The nucleocapsid (NC) protein of human immundeficiency virus type 1 (HIV-1) is necessary during virion assambly for core formation and packaging of the viral genomic RNA dimer. The NC protein of HIV-1 contains two clusters of basic aminoacids consisting of five and four aminoacid residues respectively, flanking the first of its two zinc fingers. The importance of these particular aminoacids for the binding of RNA was to be examined. METHODS: Towards this aim these aminoacid residues have been mutagenized to neutral ones individually as well as in various combinations by site-directed mutagenesis. Wild type NCp7 and the mutant proteins were expressed as recombinant proteins in E. coli with six histidines as tags at their aminotermini in order to allow efficient purification. The purified proteins were analyzed for RNA binding in vitro using HIV-1 5' leader RNA transcribed in vitro. Assays comprised North-Western blots at various salt concentrations and filter binding tests which allow determination of the dissociation constants of the various mutants. RESULTS AND CONCLUSIONS: The results indicate for the first time that mutation of the three distinct aminoacids R7, R32, and K33 are more critical for RNA binding than others. Mutation of the other aminoacid residues reduce the binding affinity proportionately to the number of mutations. Mutation of 7 out of the total of 9 basic aminoacid residues reduces the binding by 50 to 90-fold. Thus, in planning the construction of possible antiviral therapeutics the unique role of these three particular aminoacids should be considered. DE Amino Acid Sequence Amino Acids/CHEMISTRY Capsid/CHEMISTRY/*GENETICS/*METABOLISM Escherichia coli/GENETICS Gene Products, gag/CHEMISTRY/*GENETICS/*METABOLISM Human Hydrogen-Ion Concentration HIV-1/*GENETICS/*METABOLISM In Vitro Mutagenesis, Site-Directed Recombinant Proteins/CHEMISTRY/GENETICS/METABOLISM RNA, Viral/*METABOLISM Zinc Fingers/GENETICS MEETING ABSTRACT SOURCE: National Library of Medicine. NOTICE: This material may be protected by Copyright Law (Title 17, U.S.Code).