Analysis of retroviral protease cleavage sites reveals two types of cleavage sites and the structural requirements of the P1 amino acid.
Retroviruses encode a protease which cleaves the viral Gag and Gag/Pol protein precursors into mature products. To understand the target sequence specificity of the viral protease, the amino acid sequences from 46 known processing sites from 10 diverse retroviruses were compared. Sequence preference was evident in positions P4 through P3' when compared to flanking sequences. Approximately 80% of all cleavage site sequences could be grouped into two classes based on the sequence composition flanking the scissile bond. The sequences at the amino-terminal cleavage site of the major capsid protein of Gag is always a member of one of the two classes while the carboxyl-terminal cleavage site is of the other class, suggesting a biological role for the two classes. Known processing site sequences proved useful in a motif searching strategy to identify processing sites in retroviral protein sequences, particularly in Gag. In all known cleavage sites, the P1 amino acid is hydrophobic and unbranched at the beta-carbon. The sequence requirements of the P1 position were tested by site-directed mutagenesis of the P1 Phe codon in an HIV-1 Pol cleavage site. Mutations were tested for protease-mediated cleavage of the Pol precursor expressed in Escherichia coli.
Pettit
S C
SC
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Simsic
J
J
Loeb
D D
DD
Everitt
L
L
Hutchison
C A
CA
Swanstrom
R
R
D000595:Amino Acid Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism*; D015751:Genes, pol; D015497:HIV-1 / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism*; D012190:Retroviridae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D011498:Protein Precursors; D010450:Endopeptidases