SUMOylation of SARS-CoV-2 spike protein is a key target for broad-spectrum antiviral therapy

<b>Background:</b> Dynamic SUMO modifications play crucial roles in orchestrating cellular response to various stimuli, including viral infection, and hold significant therapeutic potential. The Spike (S) protein, a surface glycoprotein of SARS-CoV-2 (a global health threat), serves as the key mediator for viral entry and is a critical target for drug development. However, the function of SUMOylation in the Spike protein remains largely unclear. <b>Methods:</b> The SUMO modification of Spike was assessed by immunoprecipitation (IP), denatured IP and immunoblotting assays in lung epithelial cells or SUMO deficient cell line models. The effect of Spike SUMOylation on viral infection was explored by site-direct mutation, cell-to-cell transmission, cell-free infection, quantitative PCR and immunofluorescence staining experiments. The role of Spike SUMOylation-derived peptides was investigated using viral intranasally infection, immunohistochemistry assay in transgenic mouse model. <b>Results:</b> SARS-CoV-2 infection triggers the relocation of SUMO1 to the cytoplasm and SUMO2 to the perinuclear region. Notably, SUMO1 knockout increases Spike trimer formation and its co-localization with SUMO2 at perinuclear puncta, which facilitates virion particle release. SUMO2 knockout leads to enhanced Spike cleavage and promotes viral cell-to-cell transmission. Further bioinformatic and immuno-precipitation analyses reveal that the Spike protein contains highly conserve SUMO-interacting motifs (SIMs) and selectively promotes either SUMO1 (via SIM1) or SUMO2 (via SIM3/4) modifications on lysine residues 129 and 1269, respectively. Importantly, these modifications can be efficiently disrupted by the SIM2 motif. A cell-penetrating peptide (cpSIM2), derived from the SIM2 motif, exhibits robust and broad-spectrum inhibitory activity against SARS-CoV-2 variants infection <i>in vitro</i> and in hACE2-transgenic mice model. <b>Conclusions:</b> This study uncovers critical features of SUMOylation in regulating Spike-mediated viral spread and pathogenesis, providing a potential broad-spectrum therapeutic target for drug development against emerging SARS-CoV-2 infection.