Poster V10 - Comprehensive Identification of SUMOylated Sites of Essential Viral Protein through Engineered SUMO1/qFRET/MS Technology

Introduction: Viruses, such as SARS-CoV-2 and influenza viruses, often use host factors for their genome replication and viral particle assembly during the infections. We have found that human SUMOylation, a dynamic and reversible post-translational modification in human cells, is essential for the influenza virus life cycle. However, the essential SUMOylation site(s) remain unknown, and there is no comprehensive identification method for SUMOylation site identification. Comprehensive identification of SUMOylated sites is essential for determining the function of SUMOylation in influencing the viral proteins. However, identifications of SUMO site(s) in vivo using immunoprecipitation (IP)-Mass Spectrometry (MS) remain significant challenges due to the reversible process of SUMOylation, the presence of three human SUMO variants with different functions but similar structures and the large SUMO chain fragment that remains after trypsin digestion. To address this, we engineered different SUMO mutants to leave a short SUMO remnant (smaller than four amino acids) near the C-terminus upon digestion so that SUMO modification will be detected easily by MS. Quantitative FRET(qFRET) technology was used to validate all the comprehensive SUMOylated sites.

Materials and

Methods: To determine SUMOylation sites more accurately using MS, we engineered the SUMO1 peptides to generate smaller peptides for detection as a representation. The SUMO mutant constructions were generated by inserting cDNA in pET28b with cyan fluorescence protein. The proteins were expressed in BL21(DE3) cell lines and purified by 6x His tag to Ni-NTA beads affinity chromatography. Our unique FRET-based protein interaction assay was applied to the determinations of engineered SUMO activity and SUMOylation. Mass spectrometry data was acquired on a Thermo Orbitrap Fusion (ThermoFisher) in data-dependent mode. The LTQ-orbitrap XL raw data was analyzed using Thermofisher Proteome AnalyzerTM.

Results, Conclusions, and Discussions: We develop a novel SUMOylated site identification approach based on qFRET/MS to facilitate the simultaneous detection of SUMO modification in vitro and in vivo. We identified and confirmed three SUMOylation sites, K61, 65, 347 of SARS-CoV-2 N protein and nine SUMOylation sites of Influenza A virus M1 protein, K21, 35, 47, 57, 113, 187, 230, 242, 252 with the engineered SUMO variants using qFRET/MS-based SUMOylation assay in vitro.
These results suggest that our novel approach effectively identifies SUMOylation sites in vitro. We will continue to develop this technology to identify the SUMOylated site in vivo. Developing and characterizing different variants of SUMO through qFRET offers a new approach for identifying post-translational modification sites for viral proteins and other cellular proteins for therapeutics development in the future.

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