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).