A human immunodeficiency virus type 1 protease biosensor assay using bioluminescence resonance energy transfer

Abstract

A sensitive reporter assay to measure human immunodeficiency virus type 1 (HIV-1) protease (PR) activity is described in this manuscript. This assay measures PR activity as a function of the resonance energy transfer (RET) between a donour molecule [humanized sea pansy Renilla reniformis luciferase (hRLuc)] and an energy acceptor molecule, humanized green fluorescent protein (hGFP2) when expressed in mammalian cells. This is a naturally occurring phenomenon and is an emerging and powerful technology that has significant advantages over alternative in vitro PR assays. The HIV-1 Gag-p2/Gag-p7 (p2/p7) PR site was inserted between hGFP2 and hRLuc. The newly created vector, hRLuc-p2/p7-hGFP2 was co-expressed with an HIV-1 codon-optimized PR+ or PR− Gag/Pol expressor. Expression of the hRLuc-p2/p7-hGFP2 alone or with the PR− Gag-Pol expressor generated a BRET2 indicating that the PR cleavage site was not cleaved, whereas the inclusion of the PR+ Gag-Pol produced a significant reduction in the BRET2. The inclusion of PR inhibitors Saquinavir or Amprenavir, or the expression of a p2/p7 PR substrate mutant also blocked the cleavage to result in a stable BRET2 signal. Because the HIV-1 auxiliary protein Vif has been shown to modulate the HIV-1 p2/p7 cleavage, this assay was then validated in studies in which Vif was expressed. When Vif was overexpressed along with hRLuc-p2/p7-hGFP2 and PR+ Gag-Pol, the decrease in BRET2 was abrogated in a dose-dependent manner, demonstrating that supraphysiologic levels of Vif block p2/p7 cleavage. An accumulation of a Gag processing intermediate was observed, indicating that p2/p7 cleavage was negatively affected. Overexpression of an RNA-binding-defective Staufen protein or a related dsRNA-binding protein TRBP had no effect on PR cleavage activity as shown by Western and BRET2 analyses. The p2/p7 processing data were confirmed by Western blot analyses. BRET is non-invasive and occurs within live cells, is measured in real time, and is not restricted to cellular compartments making it an especially attractive technology to identify small bioactive inhibitory molecules. This PR BRET2 biosensor assay can be adapted for high throughput screening of new HIV-1 PR inhibitors. It can be employed to screen for antiviral compounds that also target the proteases of other viruses.