Fig. 1: SARS2-NP undergoes poly-SUMO modification.

a Scatter plot of enriched terms from gene ontology (GO) enrichment analysis of the SARS2-NP interactome in the SARS-CoV-2-infected A549 cells stably expressing hACE2 (A549-hACE2). b SUMOylation assay. Immunoblot (IB) of total lysates and anti-SARS2-NP immunoprecipitates (IP) of cell lysates from SARS-CoV-2-infected 16HBE, or A549-hACE2, or CaCo-2 cells, or lungs of hACE2-transgenic mice with SARS-CoV-2 infection. c Identification of NP SUMOylation site. Left, schematic representation of the signature tags left after trypsin digestion on peptides modified by SUMO3 WT/T90R. Right, the mean intensity (label-free quantification) of GG-K sites of SARS2-NP identified by MS/MS in SUMO3 WT/T90K-expressing HEK293T cells. d SUMOylation assay. IB of total lysates and Ni-NTA pulldown of cell lysates from HEK293T cells transfected with plasmids expressing 6His-SUMO3, plus Flag-SARS2-NP WT/K65R plasmids as indicated. e In situ PLA for HA-SUMO3 and Flag-SARS2-NP WT/K65R in HeLa cells as indicated. Left, the PLA-detected proximity (PROX) complexes are represented by the red fluorescent dots. Right, changes in PROX dots intensity. NS, not significant; ND, not determined. f IB of total lysates and anti-Flag IP from HEK293T cells transfected with indicated expression plasmids. g Aggregation assay. SDD-AGE (top) and SDS-PAGE (bottom) of lysates of HEK293T cells transfected with indicated expression plasmids. h In situ PLA for Myc-SARS2-NP, plus Flag-SARS2-NP WT/K65R in HeLa cells as indicated. Right, changes in PROX dots intensity. Data are representative of at least three independent experiments with similar results (a–h). Data are presented as Mean  ±  SD; n = 3 independent samples; Statistical analyses were performed using a two-tailed Student’s t-test (h). Scale bar, 10 μm (e, h).