Protocol for a Mammalian Cell-Based Assay for Monitoring the HIV-1 Protease Activity
互联网
592
Proteases are essential at different stages of the viral life cycle and for the establishment of a successful infection. Monitoring the catalytic activity of proteases in an easy and straightforward manner can thus drastically facilitate the discovery of novel antivirals, as well as help elucidate the activity and mechanism of action of the viral protease under study. In our laboratory, we have developed an assay in T-cells with a robust read-out to monitor the proteolytic activity of HIV-1 Protease (PR). The assay utilizes the prototypic transcription factor Gal4, which consists of the N-terminal DNA-binding domain and the C-terminal trans-activation domain. The assay is based upon (1) introduction of PR in between the two Gal4 domains to obtain a PR/Gal4 fusion protein and (2) utilization of the enhanced Green Fluorescent Protein as reporter of PR activity.
In order to overcome the possible cellular cytotoxicity of PR, the fusion protein in our assay is under the control of a tetracycline-inducible promoter. This ensures that it will be expressed only when needed, upon the addition of tetracycline or doxycycline. When active, PR has autocatalytic activity and cleaves itself from the Gal4 domains, resulting in the inability to induce eGFP expression. However, if PR activity is blocked or it is inactive, the two domains remain intact, resulting in eGFP expression. The assay can therefore be utilized to analyze the inhibitory effects of factors, peptides or compounds, designed on a rational- or nonrational-based approach, in the natural milieu of infection, where eGFP serves as a biosensor for PR activity.
