Time-Resolved Methodology for Coronavirus Non-Structural Protein Interactomics

Abstract

Protein-protein interactions govern many cellular processes including signal transduction, recognition of viral infection, and replication. Current methods to investigate protein-protein interactions capture the steady-state profile but do not encompass the timing or dynamics of these interactions. Here, we developed time-resolved analysis of protein-protein ensembles using a destabilizing domain, or TRAPPED. This methodology uses an Escherichia coli dihydrofolate reductase destabilizing domain that can be stabilized by the native ligand trimethoprim as a way to synchronize a population of a target protein for time-resolved analysis. Trimethoprim analog probes with covalent linkage and Click chemistry functionality were developed to label the synchronized population for affinity purification mass spectrometry. Using TRAPPED, we characterized the dynamic interactions of SARS-CoV and SARS-CoV-2 nonstructural protein 15 to reveal interactions with DNA-binding proteins, translation initiation machinery, and the large ribosomal subunit. In addition, steady state interactomics methods were utilized to identify putative substrates of macrodomain 1 in nonstructural protein 3 that has ADP-ribosyl hydrolase activity and has been shown to contribute to immune evasion. Members of the T-complex protein 1 ring complex were identified as interactors of macrodomain 1 suggesting a functional role of this viral domain in regulating host protein folding. Finally, with the goal of studying the dynamics of virus-host interactions during live infection, conditions for a SARS-CoV-2 replicon were optimized to increase viral protein production. Though no viral proteins were detected by bottom-up proteomics, the SARS-CoV-2 replicon can be adapted to investigate other viral functions including the strain-specific suppression of the interferon response by SARS-CoV nonstructural protein 2.