qFRET-coupled MS Technology for Identifying Essential SUMOylation Site of Influenza Virus

Introduction: : SUMOylation, a post-translational modification (PTM) of SUMO peptide conjugation to substrates, which is catalyzed by an enzyme cascade including E1 activating enzyme, E2 conjugation enzyme, and a family of E3 ligases, and play critical roles in signal transduction, cell cycle, DNA damage repair, transcription in physiology and pathological process, such as virus infections, cancers and immune responses. Influenza viruses often exploit host cellular factors to support their genome replication and viral particle assembly during infection. We have found that the influenza A virus (IAV) matrix protein M1, which is essential for viral life cycle, is SUMOylated and this SUMOyaltion is essential for infections. However, the complete set of SUMOylation sites on IAV M1 remains unknown, and no comprehensive method currently exists to detect them accurately. To address this limitation, especially the challenge posed by the large SUMO chain fragments remaining after proteolytic digestion, we developed a series of engineered SUMO1 mutants that leave a minimal C-terminal SUMO remnant, thereby improving the detection sensitivity of SUMOylated peptides using Orbitrap Fusion mass spectrometry (MS). During the process, we employed quantitative FRET (qFRET) technology to monitor the completeness of site identification.

Materials and

Methods: : To enable more accurate identification of SUMOylation sites using MS and qFRET, the engineered SUMO1 mutant constructs were developed by inserting SUMO1 cDNA fused with cyan fluorescent protein into the pET28b(+) vector. The fusion proteins were expressed in E. coli BL21(DE3) cells and purified using Ni-NTA affinity chromatography targeting the 6×His tag. SUMO activity and SUMOylation efficiency of the engineered constructs were assessed using our in-house FRET-based protein interaction assay. A549 cells were transfected with a plasmid encoding IAV M1 in the pEF1/myc-His B vector to analyze SUMOylation in a mammalian context. Mass spectrometry data were acquired using both the Thermo Orbitrap Fusion and TSQ Altis Plus systems.

Results, Conclusions, and Discussions:: Using this approach, we initially identified six SUMOylation sites on the IAV M1 protein by MS, which were then validated by qFRET. Residual FRET signals suggested the presence of additional SUMOylation sites. Upon further analysis, three additional sites were discovered. Importantly, when all nine lysine residues were mutated to arginine, the FRET signal was completely disappeared, confirming full coverage of SUMOylation sites. These nine SUMOylation sites were also confirmed in mammalian cells expressing wild-type SUMO. Our combined qFRET and MS methodology successfully mapped all SUMOylation sites on IAV M1, regardless of their abundance. These findings validate the effectiveness of our platform in detecting viral protein SUMOylation comprehensively and sensitively. In summary, we developed a novel qFRET/MS–based platform for identifying SUMOylation sites, which enabled efficient and comprehensive detection of SUMO sites modifications both in vitro and in mammalian cells. From the nine identified SUMOylation sites, key residues essential for the viral life cycle can now be investigated for further functional studies and novel therapeutics development. This technology offers a novel powerful avenue for the site identification of SUMOyaltion and other Ubiquitin-like modifications.

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