A high throughput multiplexed platform for monitoring proteolysis in the classical secretory pathway -- Search for novel antivirals.

摘要

RNA Viruses such as Human Immunodeficiency (HIV) and Dengue Virus (DenV) cause devastating diseases, including Acquired Immunodeficiency Syndrome (AIDS) and Dengue Hemorrhagic Fever. With 2.5 and 50 million new infections yearly, respectively, unsuccessful attempts at vaccine development and resistance to current treatment, all beg for novel antivirals. However, new methods and platforms are required to increase the chances for their discovery.;The classical secretory pathway is essential for biological functions and is utilized for the transport of proteins to the cell surface and/or extracellular matrix. Within the secretory pathway resides an array of enzymes that modify proteins into their mature and active forms. Viruses, as well as other pathogens, hijack such enzymes for their own benefit. While HIV and DenV are distant viruses, both rely on the cellular protease Furin within this compartment for maturation of their viral envelopes. Importantly, blockade of Furin processing leads to non-fusogenic virions, making it an ideal drug target.;In order to monitor proteolysis within the natural milieu of the secretory pathway, an assay with a robust and quantitative read-out was developed. The assay relies on a complex scaffold molecule targeted to the Endoplasmic Reticulum for transport to the cell surface. A substrate is flanked by the FLAG and HA tags fused to the N-terminus of the scaffold. In such a way, in the absence of proteolysis both tags are presented on the cell surface but only one tag (HA) is presented if proteolysis occurs. Tag presentation can be subsequently detected by fluorescent-coupled antibodies in a robust and quantitative manner through flow cytometry and/or microscopy techniques.;The assay described has been adapted to monitor processing of the HIV envelope and DenV pr-M proteins. It has been further calibrated in a 96-well plate format for flow cytometry, to demonstrate both robustness and repeatability, critical for high throughput screening. In addition, varied intensities of a stably expressed fluorescent protein were used for genetic barcoding through retroviral technology. Genetic barcoding was exploited to further develop the platform in a multiplexed format to assay multiple substrates in the same well. A robust platform such as the one described here will expand high throughput capabilities in search for novel antivirals and can additionally be adapted to other substrates in the secretory pathway.