An HIV-1 protease screening assay using a non-infectious proviral clone.
An in-vitro assay was developed to screen for HIV-1 protease inhibitors using a non-infectious proviral clone (X19) with a deletion in the envelope gene (Ratner et al., 1991). The proviral clone, X19, was expressed transiently in the COS 7 cell line. The virus was able to replicate as assessed by the presence of p24 in the supernatants, yet the virions produced did not infect CD4 positive cells. To determine the effect of a known protease inhibitor on p24 antigen production, PD 148310 (a Ro 31-8959 analog) was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 h and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. PD 148310 inhibited p24 release in a dose-dependent manner with an IC50 of 23.6 nM. Western blot analysis of the supernatants using a mouse monoclonal antibody against p24 confirmed the presence of a well-defined p24 band in the control lane. At 1000 nM of PD 148310 the p24 band was not detectable, leaving only the unprocessed p55 Gag precursor. This technique is a useful tool to screen for potential HIV-1 protease inhibitors.
Heldsinger
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Department of Infectious Diseases, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI 48105.
Antonucci
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D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017353:Gene Deletion; D015683:Gene Products, gag / Q000378:metabolism; D015752:Genes, env; D016655:HIV Core Protein p24 / Q000096:biosynthesis; D016333:HIV Protease / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D008826:Microbial Sensitivity Tests / Q000379:methods*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011533:Proviruses / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D014162:Transfection; D015215:Zidovudine / Q000494:pharmacology
D015683:Gene Products, gag; D016655:HIV Core Protein p24; D017320:HIV Protease Inhibitors; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D015215:Zidovudine; D016333:HIV Protease