       Document 0648
 DOCN  M9640648
 TI    Mutations of basic amino acids of NCp7 of human immunodeficiency virus
       type 1 affect RNA binding in vitro.
 DT    9604
 AU    Schmalzbauer E; Strack B; Dannull J; Guehmann S; Moelling K; Institut
       fur Medizinische Virologie, Universitat Zurich,; Switzerland.
 SO    J Virol. 1996 Feb;70(2):771-7. Unique Identifier : AIDSLINE MED/96135185
 AB    The nucleocapsid (NC) protein of human immunodeficiency virus type 1 is
       required for packaging of viral RNA and for virion assembly. It contains
       two clusters of basic amino acids, consisting of five and four amino
       acid residues, flanking the first of its two zinc fingers. These amino
       acid 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
       Escherichia coli, with six histidines as tags at their amino termini in
       order to allow efficient purification. The purified proteins were
       analyzed for RNA binding in vitro with human immunodeficiency virus type
       1 5' leader RNA transcribed in vitro. Assays comprised Northwestern
       blots at various salt concentrations and filter binding tests which
       allowed determination of the dissociation constants of the various
       mutants. The results indicated that mutations of the amino acid R-7 and
       of R-32 and K-33 were more critical for RNA binding than other
       mutations. Mutation of the other amino acid residues reduced the binding
       affinity in proportion to the number of mutations. Mutation of seven of
       the nine basic amino acid residues reduced the binding of RNA by 50- to
       90-fold.
 DE    Amino Acid Sequence  Base Sequence  Blotting, Northern  Blotting,
       Western  Capsid/GENETICS/ISOLATION & PURIF/*METABOLISM  Cloning,
       Molecular  DNA, Viral  Gene Products, gag/GENETICS/ISOLATION &
       PURIF/*METABOLISM  Human  HIV-1/*METABOLISM  Molecular Sequence Data
       Mutation  Recombinant Fusion Proteins/GENETICS/ISOLATION &
       PURIF/METABOLISM  RNA, Viral/*METABOLISM  Structure-Activity
       Relationship  Support, Non-U.S. Gov't  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

