Development of FRET and Stress Granule Dual-Based System to Screen for Viral 3C Protease Inhibitors

Abstract

3C proteases (3Cpros) of picornaviruses and 3C-like proteases (3CLpros) of coronaviruses and caliciviruses represent a group of structurally and functionally related viral proteases that play pleiotropic roles in supporting the viral life cycle and subverting host antiviral responses. The design and screening for 3C/3CLpro inhibitors may contribute to the development broad-spectrum antiviral therapeutics against viral diseases related to these three families. However, current screening strategies cannot simultaneously assess a compound’s cytotoxicity and its impact on enzymatic activity and protease-mediated physiological processes. The viral induction of stress granules (SGs) in host cells acts as an important antiviral stress response by blocking viral translation and stimulating the host immune response. Most of these viruses have evolved 3C/3CLpro-mediated cleavage of SG core protein G3BP1 to counteract SG formation and disrupt the host defense. Yet, there are no SG-based strategies screening for 3C/3CLpro inhibitors. Here, we developed a fluorescence resonance energy transfer (FRET) and SG dual-based system to screen for 3C/3CLpro inhibitors in living cells. We took advantage of FRET to evaluate the protease activity of poliovirus (PV) 3Cpro and live-monitor cellular SG dynamics to cross-verify its effect on the host antiviral response. Our drug screen uncovered a novel role of Telaprevir and Trifluridine as inhibitors of PV 3Cpro. Moreover, Telaprevir and Trifluridine also modulated 3Cpro-mediated physiological processes, including the cleavage of host proteins, inhibition of the innate immune response, and consequent facilitation of viral replication. Taken together, the FRET and SG dual-based system exhibits a promising potential in the screening for inhibitors of viral proteases that cleave G3BP1.

Stress Granule Core Protein-Derived Peptides Inhibit Assembly of Stress Granules and Improve Sorafenib Sensitivity in Cancer Cells

“…Immunofluorescence staining was performed as described previously [53]. HeLa cells were fixed in 4% paraformaldehyde for 30 min at 4 °C and washed with phosphate buffered saline (PBS) three times.…”

Development of FRET Biosensor to Characterize CSK Subcellular Regulation

“…Therefore, the EIY version was chosen for subsequent research in this work. 25,20,19,22,23). In cells transfected with Src-tail, Fyn-tail, and EIY CSK-FRET (1.0 µg of DNA on a 24-well plate), the ECFP/FRET ratio values (mean ± S.D.)…”

“…Although membrane localization is important in the regulation of CSK's function, there is a lack of imaging tools that can directly visualize CSK's activity at the plasma membrane and its submembrane microdomains. Fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide powerful tools for the monitoring of biochemical activity and visualization of signaling molecules in vitro and in living cells [21][22][23]. The FRET donor and acceptor pair, consisting of enhanced cyan fluorescent protein (ECFP) and a variant of yellow fluorescent protein (YPet), has been optimized for FRET biosensor engineering; it can form weak dimers that enhance the efficiency and sensitivity of FRET [24].…”

Picornavirus 3C Proteins Intervene in Host Cell Processes through Proteolysis and Interactions with RNA

“…Host cells utilize a variety of antiviral mechanisms to combat picornavirus infection, including stress granule formation, innate inflammatory responses, autophagy, and a few others [2,125,134]. Picornaviruses must attack the cellular proteins engaged in these antiviral defenses to survive, and 3C is essential to these processes [87][88][89].…”