Development of Tools and Proteomic Strategy to Elucidate the Role of SUMOylation at the Intersection of SARS-CoV-2 Replication and Host Cellular Pathways

To elucidate how SARS-CoV-2 infection modulates global SUMOylation and whether SARS-CoV-2 viral proteins are SUMOylated, we developed a complete pipeline including two novel and robust cellular models A549-ACE26xHis-SUMO2 and A549ACE26xHis SUMIO2AA that stably express 6xHis-SUMO2 and are susceptible to SARSCoV-2 infection. The cellular models containing the exogenous 6xHis-SUMO2 can conjugate to target proteins and allows for the detection of SUMO-2 modified proteins. We implemented the Ni2+NTA affinity purification system to successfully capture, enrich and purify 6xHis-SUMO2 conjugates. We successfully trypsin digested purified 6xHis-SUMO2 conjugates by coupling on-beads digestion with the Ni2+NTA resin which has not been reported before and utilised LC-MS/MS and Labelled Free Quantification (LFQ) to characterise 6xHis-SUMO2 conjugates. Our proteomic strategy identified 67 statistically significant and enriched SUMOylated proteins which included proteins part of the SUMOylation pathway like UBC9, UBA2 and TRIM28 as well as known SUMO-2 cellular targets like PML, SP100 and RANGAP1. This pipeline can be utilised in the future to identify how SARS-CoV-2 modulates global SUMOylation and whether SARS-CoV-2 proteins are targets for SUMOylation.