Fluorescent genetic bar-coding for biological applications : adaptation for monitoring HIV-1 PR variants in mammalian cells

Includes bibliographical references (pages 31-35).Human Immunodeficiency Virus (HIV) is the causal agent of Acquired Immunodeficiency Syndrome (AIDS), a pandemic responsible for approximately two million deaths each year. HIV remains a true world health concern despite progress in the field of antiviral therapy, mainly due to emerging drug resistant viruses. Thus, innovative methods are needed to facilitate the discovery of novel antivirals. Cell based assays utilizing retroviral technology allows for the stable expression of genes of interest in a wide range of eukaryotic cells. We have previously developed an assay where the HIV-1 PR is fused between the DNA-binding and transactivation domains of the Gal4 prototypic transcription factor. PR fused within Gal4 auto-catalytically cleaves itself, leaving behind two non-functional Gal4 domains. However, when inhibited, the fusion protein remains intact, retaining its function as a transcription factor. The intact Gal4-PR fusion activates expression of the green fluorescence reporter gene, which serves as a biosensor for PR activity. The assay was adapted to drug resistant PR variants in a multiplex format for flow cytometry. We exploited the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins through retroviral technology. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. The multiplexed format enhances high throughput capabilities for screening of peptides and chemical compound libraries and serves as a platform for drug discovery against PRs of important human viral pathogen