An in-vivo protease inhibitor screening assay using a non-infectious HIV-1 proviral clone.

Important note: Information in this article was accurate in 1993. The state of the art may have changed since the publication date.

An in-vivo protease inhibitor screening assay using a non-infectious HIV-1 proviral clone.

Abstr Gen Meet Am Soc Microbiol. 1993;93:434 (abstract no. T-16). Unique Identifier : AIDSLINE ASM93/93291812
Heldsinger AA; Antonucci TK; Department of Infectious Diseases, Parke-Davis Pharmaceutical; Research, Division of Warner Lambert Company, Ann Arbor, MI; 48105.

Abstract: We developed an in-vivo assay to screen for protease inhibitors using a non-infectious HIV-1 proviral clone (X19) with a deletion in the envelope gene. COS 7 cells were transfected by the calcium phosphate technique with 10 micrograms of X19 DNA cloned into the plasmid pSP65. To control for variation in the transfection efficiency the cells were co-transfected with a plasmid DNA, pCMVlacz, which has the reporter gene beta galactosidase under control of the CMV promoter. To determine the effect of a known protease inhibitor Ro 31-8959 on p24 antigen production, the inhibitor was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 hours and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. Ro 31-8959 inhibited p24 release in a dose dependent manner with a IC50 of 14 nM. A Western blot analysis of the supernatants using a p24 mouse monoclonal antibody confirmed the presence of a well-defined p24 band in the control lane. At 1000nM RO 31-8959 the p24 band was not detectable, revealing only unprocessed p55 gag precursor. This technique is now in use to screen for potential HIV-1 protease inhibitors.

Keywords: beta-Galactosidase/GENETICS/METABOLISM Animal Cell Line Enzyme-Linked Immunosorbent Assay HIV Core Protein p24/ANALYSIS/*BIOSYNTHESIS HIV Protease Inhibitors/*PHARMACOLOGY HIV-1/*DRUG EFFECTS/GROWTH & DEVELOPMENT/METABOLISM Isoquinolines/*PHARMACOLOGY Microbial Sensitivity Tests/*METHODS Promoter Regions (Genetics) Proviruses/*DRUG EFFECTS/GROWTH & DEVELOPMENT Quinolines/*PHARMACOLOGY Transfection ABSTRACT
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