Mutations of basic amino acids of NCp7 of human immunodeficiency virus type 1 affect RNA binding in vitro.
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.
Schmalzbauer
E
E
Institut für Medizinische Virologie, Universität Zürich, Switzerland.
Strack
B
B
Dannull
J
J
Guehmann
S
S
Moelling
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002213:Capsid / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D036022:Capsid Proteins; D003001:Cloning, Molecular; D004279:DNA, Viral; D015683:Gene Products, gag / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus