(D-151) Discover Essential Host SUMOyaltion Pathway for Virus Life Cycle Through qFRET/MS-based Technology

Introduction:: As a pathogen to humans, viruses often take advantage of the host factors for their infection and replication. Identifying and elucidating essential host pathways for viral infection is critical for understanding the viral infection processes and developing novel therapeutics with broad-spectrum treatment modalities for anti-viruses, such as IAV/IBV and COVID-19.


Materials and Methods:: An in vitro qFRET-based SUMOylation reaction was conducted in the presence of CyPet-SUMO1, YPet‑IAV-M1, E1 hetero-dimer AOS1/UBA2, E2 conjugating enzyme UBC9, E3 ligase PIAS1. After a FRET measurement to make sure the M1 was SUMOylated, the conjugated substrate was digested with Pierce^TM Glu-C Protease before the SUMOylated Lys sites were determined using Thermo Orbitrap Fusion (ThermoFisher). Five Lys sites of M1, K21/35/57/230/242 were identified. To determine the functional role of these potential SUMOylation sites, each of these identified Lys site or in combination were then mutated to Arg in the IAV M1 gene for virus reconstitution. The reconstitute IAV virus supernatants were then examined for virus particles using a viral plaque assay. In another functional assay of SUMOylation in the IAV viral life cycle, viral plaque assays were performed in the presence or absence of a novel SUMOylation-specific inhibitor, STE025, discovered from our qFRET-based high-throughput screening.


Results, Conclusions, and Discussions:: Results and Discussion: The human SUMOylation pathway was identified as an essential pathway for IAV/IBV life cycle, and a critical SUMOylation site in virus M1 capsid protein was identified. Inhibition of human SUMOylation using a novel SUMOylation inhibitor discovered from our qFRET-based HTS inhibited the virus life cycle, and no drug resistance was developed against the essential host pathway in both IAV/IBV.

Conclusions: An essential host pathway, human SUMOylation, has been discovered for IAV/IBV life cycle. The requirement of the human SUMOylation pathway for viruses is elucidated on the SUMOylation site of the IAV/IBV M1 protein.

Discussion: A host pathway is demonstrated as essential for the virus life cycle for the first time. The blockage of the essential host pathway for viruses could be a novel strategy for future anti-virus therapeutics with a broad spectrum and no drug resistance development.


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