In vitro processing of HIV-1 nucleocapsid protein by the viral proteinase: effects of amino acid substitutions at the scissile bond in the proximal zinc finger sequence.
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein flanked by Gag sequences (r-preNC) was expressed in Escherichia coli and purified. HIV-1 proteinase cleaved r-preNC to the "mature" NCp7 form, which is comprised of 55 residues. Further incubation resulted in cleavages of NCp7 itself between Phe16 and Asn17 of the proximal zinc finger domain and between Cys49 and Thr50 in the C-terminal part. Kinetic parameters determined for the cleavage of oligopeptides corresponding to the cleavage sites in r-preNC correlated well with the sequential processing of r-preNC. Mutations of Asn17 were introduced to alter the susceptibility of NC protein to HIV-1 proteinase. While mutating Asn17 to Ala resulted in a protein which was processed in a manner similar to that of the wild type, mutating it to Phe or Leu resulted in proteins which were processed at a substantially higher rate at this site than the wild type. Mutation of Asn17 to Lys or Gly resulted in proteins which were very poorly cleaved at this site. Oligopeptides containing the same amino acid substitutions at the cleavage site of the proximal zinc finger domain were also tested as substrates of the proteinase, and the kinetic parameters agreed well with the semiquantitative results obtained with the protein substrates.
Tözsér
József
J
National Cancer Institute, Frederick, Maryland 21701, USA.
Shulenin
Sergey
S
Louis
John M
JM
Copeland
Terry D
TD
Oroszlan
Stephen
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009842:Oligopeptides; D011498:Protein Precursors; D011994:Recombinant Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease