Cleavage of HIV-1 gag polyprotein synthesized in vitro: sequential cleavage by the viral protease.
The virally encoded protease of human immunodeficiency virus is responsible for the processing of the gag and gag-pol polyprotein precursors to their mature polypeptides. Since correct processing of the viral polypeptides is essential for the production of infectious virus, HIV protease represents a potential target for therapeutic agents that may prove beneficial in the treatment of AIDS. In this study, full-length gag polyprotein has been synthesized in vitro to serve as a substrate for bacterially expressed HIV-1 protease. Expression of the protease in E. coli from the lac promoter was enhanced approximately five-fold by deletion of a potential hairpin loop upstream from the codon determining the amino terminus of mature protease. Extracts of induced cultures of E. coli harboring a protease-containing plasmid served as the source of protease activity. The gag polyprotein synthesized in vitro was cleaved by such lysates, producing fragments corresponding in size to p17 plus p24 and mature p24. Immunoprecipitations with monoclonal antibodies to p17 and p24 polypeptides suggest that initial cleavage of gag polyprotein occurs near the p24-p15 junction. The proteolysis was inhibited by pepstatin with an IC50 of 0.15 mM for cleavage at the p24-p15 junction and 0.02 mM for cleavage at the p17-p24 junction.
Erickson-Viitanen
S
S
Medical Products Department, E.I. duPont de Nemours and Co., Inc., Wilmington, DE 19898.
Manfredi
J
J
Viitanen
P
P
Tribe
D E
DE
Tritch
R
R
Hutchison
C A
CA
Loeb
D D
DD
Swanstrom
R
R
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D004279:DNA, Viral / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D005822:Genetic Vectors; D016333:HIV Protease; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D010957:Plasmids; D011499:Protein Processing, Post-Translational
D004279:DNA, Viral; D015683:Gene Products, gag; D010436:Pepstatins; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016333:HIV Protease; C031375:pepstatin