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. 1993 Nov 30;134(1):123-8.
doi: 10.1016/0378-1119(93)90185-6.

Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity

M G Murray  1 , W Hung, I Sadowski, B Das Mahapatra
Affiliations

Inactivation of a yeast transactivator by the fused HIV-1 proteinase: a simple assay for inhibitors of the viral enzyme activity

M G Murray et al. Gene. .

Abstract

The human immunodeficiency virus type 1 (HIV-1) proteinase (PR) and its flanking sequences have been fused in frame between the DNA-binding domain and the transcription-activation domain of the yeast protein, GAL4. As has been shown before with the 3C proteinase of Coxsackie virus B3 (CVB3) [Das Mahapatra et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4159-4162], the GAL4::PR fusion protein retains its GAL4 function, providing the PR is inactive. When PR is active, its autocatalytic activity in the hybrid protein is shown to inactivate the transactivation function of GAL4. This provides a simple assay to monitor PR activity. A dose-dependent effect of a potent PR-specific inhibitor is demonstrated in this system and illustrates the sensitivity of the assay. The assay is used for high throughput screening to identify novel inhibitors of the viral PR, and provides a method to generate and analyze mutants and revertants of the PR.

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