In Vitro Reconstitution and Analysis of SARS-CoV-2/Host Protein–Protein Interactions

Abstract

The emergence of viral threats such as Ebola, ZIKA, and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) requires a rapid and efficient approach for elucidating mechanisms of pathogenesis and development of therapeutics. In this context, cell-free protein synthesis (CFPS) holds a promise to resolve the bottlenecks of multiplexed protein production and interaction analysis among host and pathogen proteins. Here, we applied a eukaryotic CFPS system based on Leishmania tarentolae extract (LTE) protein expression in combination with AlphaLISA proximity-based protein interaction technology to identify intraviral and viral-human protein interactions of SARS-CoV-2 virus that can potentially be targeted by the existing or novel antiviral therapeutics. We produced and tested 54 putative human-viral protein pairs in vitro and identified 45 direct binary protein interactions. As a casing example of the assay's suitability for drug development applications, we analyzed the effect of a putative biologic on the human angiotensin-converting enzyme 2/receptor-binding domain (hACE2/RBD) interaction. This suggests that the presented pathogen characterization platform can facilitate the development of new therapeutic agents.

Exploring Viral–Host Protein Interactions as Antiviral Therapies: A Computational Perspective

“…Viral–host interactions can be characterized using three major experimental methods: yeast two-hybrid assays, co-immunoprecipitation, and mass spectrometry [ 54 , 55 , 56 , 57 ]. Yeast two-hybrid assays facilitate high-throughput screening and mimic in vivo conditions, making them valuable for detecting weak or transient interactions.…”

Cell-free protein synthesis for nonribosomal peptide synthetic biology

“…Another prominent CFPS system derived from eukaryotes developed by Alexandrov and coworkers harnesses the nonpathogenic single-celled flagellate Leishmania tarentolae (Mureev et al, 2009;Kovtun et al, 2011;Johnston and Alexandrov, 2014;Gagoski et al, 2015), which is inexpensive in bioreactors and has been successful in expressing a range of complex eukaryotic proteins (Mureev et al, 2009;Kovtun et al, 2010;Gagoski et al, 2017;Wu et al, 2022;Moradi et al, 2023). Leishmania tarentolae can be fermented on a large scale and its genetic modification process is not complicated (Kovtun et al, 2010).…”