A Novel Two-Tag System for Monitoring Transport and Cleavage through the Classical Secretory Pathway – Adaptation to HIV Envelope Processing

Abstract

The classical secretory pathway is essential for the transport of a host of proteins to the cell surface and/or extracellular matrix. While the pathway is well-established, many factors still remain to be elucidated. One of the most relevant biological processes that occur during transport involves the cleavage of pro-proteins by enzymes residing in the endoplasmic reticulum/Golgi/TransGolgi Network compartment. Teasing out the requirements involved in the classical secretory pathway and cleavage during transport would shed new light into mis-regulation leading to disease. Current methodologies fail to link transport and cleavage at the single cell level. Here, we describe a cell-based assay that relies on an engineered protein scaffold that can discriminate between transport to the cell surface, in the absence or presence of cleavage. Our novel two-tag system works in a robust and quantitative manner and distinguishes between cleaved and non-cleaved events based on cell surface expression of one or two epitope tags, respectively. Here, we have used the HIV-1 envelope as a substrate, which is cleaved during transport, as proof of principle. Importantly, this assay can be easily coupled to existing siRNA-based screens to identify novel regulators and effectors involved in transport and/or cleavage of cell surface proteins. In addition, unlike other in vivo based assays, the assay described here can also be easily adapted to drug discovery purposes.

A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus

“… 24 The well-established Furin substrate of HIV Env was exploited for that purpose, while serving also as control for cleavage specificity. 15 , 21 , 28 – 33 A pilot screen of 1280 small molecules contained in the Prestwick Chemical Library (PCL) was conducted using our established multiplexed assay and revealed putative hits, demonstrating the high-throughput multiplexed capabilities of our novel cell-based assay for drug discovery against DenV.…”

“…We have previously described an assay that relies on a two-tag system to monitor the processing of the human immunodeficiency virus (HIV) envelope (Env) protein. 21 Here, we describe for the first time the adaptation of the assay for the monitoring of DenV prM processing. The assay uses the expression of an engineered scaffold containing a putative substrate, the DenV prM boundary, flanked by two epitope tags, FLAG and HA.…”

“… 22 , 23 Our two-tag system can specifically couple the observed phenotype with a specific cell within a population, allowing for multiplexing in a robust and reliable manner. 21 , 24 For the first time, we have developed a novel, high-throughput, multiplexed, cell-based platform that relies on an antibody staining-based approach that can be readily exploited to analyze living cells in the presence of peptide or chemical compound libraries. In addition, although platforms for the discovery of DenV antivirals have been established, 5 , 25 – 27 no such platform exists that specifically targets prM maturation while mimicking not only a cellular context but also, more specifically, the cellular compartment where prM cleavage occurs.…”

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

“…Panels of cell populations distinguishable based on their fluorescent characteristics enhance multiplexed capabilities, which can be further exploited in combination with cell-based assays that tackle different biological questions. The protocol also describes how to engineer a panel of barcoded cells bearing one of the cell-based assays previously developed in the laboratory, as example 59 . This protocol is thus not intended to show the well-established retroviral/lentiviral technology for genetic transfer, the value of fluorescent proteins or the applications of flow cytometry 60,48 but rather to show the enhancing power of combining the three for multiplexed applications.…”

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry

“…We have genetically barcoded cells with different fluorescent proteins, tested their stability across multiple generations, and obtained distinct clonal populations based on differential fluorescent intensities. Moreover, to demonstrate biological applications, established genetically barcoded cells have been adapted to two of our existing cell‐based assays . The coupling of genetically barcoded cells to cell‐based assays will enhance high‐throughput capabilities by reducing the number of screens needed.…”