Abstract
The emergence of resistant HIV strains, together with the severe side-effects of existing drugs and lack of development of effective anti-HIV vaccines highlight the need for novel antivirals, as well as innovative methods to facilitate their discovery. Here, we have developed an assay in T-cells to monitor the proteolytic activity of the HIV-1 protease (PR). The assay is based on the inducible expression of HIV-1 PR fused within the Gal4 DNA-binding and transactivation domains. The fusion protein binds to the Gal4 responsive element and activates the downstream reporter, enhanced green fluorescent protein (eGFP) gene only in the presence of an effective PR Inhibitor (PI). Thus, in this assay, eGFP acts as a biosensor of PR activity, making it ideal for flow cytometry based screening. Furthermore, the assay was developed using retroviral technology in T-cells, thus providing an ideal environment for the screening of potential novel PIs in a cell-type that represents the natural milieu of HIV infection. Clones with the highest sensitivity, and robust, reliable and reproducible reporter activity, were selected. The assay is easily adaptable to other PR variants, a multiplex platform, as well as to high-throughput plate reader based assays and will greatly facilitate the search for novel peptide and chemical compound based PIs in T-cells.
Highlights
The Human Immunodeficiency Virus (HIV) was identified as the causative agent of Acquired Immuno Deficiency Syndrome (AIDS) in 1983 [1] and has since resulted in over 33 million deaths (UNAIDS/WHO Report 2009)
A reporter vector, referred to as pFR-enhanced green fluorescent protein (eGFP), containing a 5xUAS Gal4 responsive element upstream of a minimal CMV promoter followed by the eGFP gene (Fig. 2A) was transiently expressed in adherent HEK293T cells and these cells showed little to no background (Figs. 2B and 2C)
Even though other variants of Gal4, such as Gal4VP16, are capable of higher levels of induction under the Upstream Activation Sequences (UAS) control, minimal Gal4 allows for insertion of a proteolytic enzyme within its well-characterized domains, while still retaining high level of transcriptional activity
Summary
The Human Immunodeficiency Virus (HIV) was identified as the causative agent of Acquired Immuno Deficiency Syndrome (AIDS) in 1983 [1] and has since resulted in over 33 million deaths (UNAIDS/WHO Report 2009). In spite of the lack of progress in the field of vaccine development against HIV, three decades of research has resulted in various antivirals. The antiviral drugs developed so far target the viral proteins, including Protease (PR), Reverse Transcriptase, Integrase and Envelope (fusion process), or the cellular receptors involved in viral entry. Inhibitors, supplied as a cocktail of drugs of three or more inhibitors through Highly Active Anti-Retroviral Therapy (HAART) have resulted in a drastic reduction in the number of AIDS-related deaths [5]
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