Inhibition of bacterially expressed HIV protease activity determined by an in vitro cleavage assay with MuLV Pr65gag.
HIV protease is a virally coded enzyme that cleaves gag as well as gag-pol precursor polyproteins into functional products needed for virus assembly. A pUC plasmid containing an HIV insert starting at the 5' end of the pol gene and ending just inside the intergrase coding sequence was expressed in E. coli. It provided an 11 kD gene product (protease) that specifically cleaved the Gazdar MuLV Pr65gag precursor into Pr40gag (p30 + p10) and Pr27gag (p15 + p12) intermediates, as well as lower molecular weight gag-encoded products. These were detected by immunoblotting with either MuLV anti-p30 or p12 sera. Using cleavage of MuLV Pr65gag as an assay system, pepstatin A, fusidic acid, and cerulenin were observed to inhibit HIV protease cleavage by greater than 50% at concentrations of 0.1, 0.2-0.5, and 0.5 mM, respectively.
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Department of Microbiology, Immunology and Parasitology, LSU Medical Center, New Orleans.
Oroszlan
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Luftig
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D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015683:Gene Products, gag; D005814:Genes, Viral; D006678:HIV / Q000201:enzymology* / Q000235:genetics; D016333:HIV Protease; D015151:Immunoblotting; D009052:Leukemia Virus, Murine / Q000235:genetics; D011480:Protease Inhibitors; D012191:Retroviridae Proteins / Q000235:genetics
D015683:Gene Products, gag; D011480:Protease Inhibitors; D012191:Retroviridae Proteins; D010450:Endopeptidases; D016333:HIV Protease