Mutagenesis of protease cleavage sites in the human immunodeficiency virus type 1 gag polyprotein.
The virally encoded protease of human immunodeficiency virus (HIV) is responsible for specific cleavage events leading to the liberation of the enzymes reverse transcriptase, integrase, ribonuclease H, and the core proteins from the gag-pol and gag polyprotein precursors. Utilizing gag polyprotein synthesized in vitro, we have shown that this substrate is sequentially cleaved by purified HIV protease to yield products that on the basis of their sizes and immunoreactivities correspond to p15, p6, p7, p17, and finally mature p24. We have placed unique restriction sites flanking the p17-p24 domain in order to facilitate replacement of cleavage site sequences by utilizing oligonucleotide cassettes. Replacement of the rapidly cleaved methionine-methionine bond at the p24-p15 junction with tyrosine-proline or replacement of the tyrosine-proline bond at the p17-p24 junction with methionine-methionine results in sites that cannot be efficiently cleaved. A basic amino acid at the p17-p24 scissile bond is not tolerated. Replacement of this cleavage site with an inverted repeat amino acid sequence gives intermediate rates of cleavage. In an attempt to convert the p17-p24 domain into a p24-p15 domain, residues flanking the scissile bond were exchanged in an expanding iterative fashion. When four residues flanking the scissile bond had been replaced, the rate of cleavage relative to that of the native p17-p24 sequence was increased fourfold. The cleavage rate of the native p24-p15 sequence is still some 10-fold greater than that of the p17-p24 sequence, suggesting that more-distant residues significantly affect the cleavage rate.
Tritch
R J
RJ
Medical Products, Department, E. I. DuPont de Nemours and Co., Inc., Wilmington, Delaware 19880-0400.
Cheng
Y E
YE
Yin
F H
FH
Erickson-Viitanen
S
S
D000595:Amino Acid Sequence; D002460:Cell Line; D015683:Gene Products, gag / Q000235:genetics* / Q000378:metabolism; D016333:HIV Protease / Q000235:genetics* / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015183:Restriction Mapping; D013379:Substrate Specificity
D015683:Gene Products, gag; D016333:HIV Protease