Fig 2: PIAS1 mediates robust SUMOylation of IAV PB2 and has no effect on IAV PB1 SUMOylation.(A-C) PIAS1 catalyzes SUMOylation of IAV PB2 with SUMO1 (A), SUMO2 (B), and SUMO3 (C). HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB2, along with or without Flag-SUMO1 (A), Flag-SUMO2 (B), Flag-SUMO3 (C), Ubc9-V5, Myc-PIAS1, and Myc-PIAS1 mutants. HEK293T cells transfected to express only Flag-SUMO1/2/3 and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a mouse anti-HA mAb, and were then subjected to western blotting with a rabbit anti-PB2 pAb and a mouse anti-Flag mAb for the detection of PB2 and SUMO1 (A), SUMO2 (B), and SUMO3 (C), respectively. (D) PIAS1 has no effect on the SUMOylation of IAV PB1. HEK293T cells were transfected with plasmids expressing HA-tagged WSNPB1, along with or without Flag-SUMO1, Ubc9-V5, Myc-PIAS1, and Myc-PIAS1 mutants. HEK293T cells transfected to express only Flag-SUMO1 and Myc-PIAS1 served as a negative control. At 36 h post-transfection, cell lysates were immunoprecipitated with a rabbit anti-HA pAb, and were then subjected to western blotting with a mouse anti-PB1 mAb and a mouse anti-Flag mAb for the detection of PB1 and SUMO1, respectively. Red triangle indicates the corresponding SUMOylated viral protein.

Fig 3: MCM10-rs2274110[A] variant stables the expression of MCM10 protein by increasing its SUMOylation level. (A and B) The mRNA (A) and protein expression (B) levels of MCM10 were detected by qRT–PCR and western-blotting assays, respectively, in KYSE30 and KYSE150 cells. Cells were seeded in six-well plates after transfection with the pcDNA-Ctrl, MCM10[A], and MCM10[G]. All qRT-PCR data are presented as the mean ± SD from three repeated experiments, each with triplicates. **p < 0.01 were calculated using a two-sided Student's t-test. (C) Post-translational modifications prediction of variant rs2274110 using the PhosphoSitePlus database (https://www.phosphosite.org/homeAction) revealing that rs2274110[A] > [G] causes a Lys[K]699Arg[R] change and disrupt a SUMOylation site at K669 of MCM10. (D) Proteomic screening by mass spectrometry (MS) for the identification of SUMOylation site of MCM10. p < 0.05 was selected as the threshold of significance. (E) Co-IP analysis using anti-SUMO2/3 or anti-MCM10 antibodies. Cells were seeded in six-well plates after transfection with the pcDNA-Ctrl, MCM10[A] and MCM10[G]. (F) Western blotting analysis revealing the effect of SUMO2/3 on MCM10 protein expression. Cells were seeded in six-well plates after transfection with the siRNAs targeting SUMO2/3 or siControl. Then, cells were transfected with MCM10[A], MCM10[G], or the control vector. (G and H) The degradation of MCM10 was detected by western blotting (Upper) and quantitative analysis in the presence of CHX (Below). MCM10[A] or MCM10[G] was transfected into KYSE30 (G) and KYSE150 (H) cells treated with translation inhibitor Cycloheximide (CHX, 100 µg/ml) for 0, 2, 4, 6, 8, and 10 h. **p < 0.01 values were calculated using a two-sided Student's t-test

Fig 4: HIV-1 induces proteasome-independent loss of cellular SUMO conjugates.(A, B) HEK293 cells (A) or CD4+ Jurkat T lymphocytes (B) were transfected or electroporated with the lentiviral vector pfNL43-dE-EGFP encoding the HIV-1 genome as described above, then treated for 24 h with 3 µM MG132, a proteasome inhibitor drug, before lysis (at indicated times post-transfection/electroporation). Global SUMO1 and SUMO2/3 conjugates were analyzed by Western blot. Densitometric quantifications of SUMO signals were normalized to actin expression (which serves as a loading control). Data are presented as mean ± SEM (n = 5 for HEK293 cells, n = 4 for Jurkat cells), asterisks denote statistical significance (P-values were calculated using t test assuming unequal variances, ns: not significant). Control: cells transfected with an empty vector backbone devoid of HIV-1 genes, HIV: cells expressing the HIV-1 genome. Ubiquitin blots are shown in Fig S2.

Fig 5: HIV-1 diminishes the abundance of cellular SUMO conjugates.(A, B) A lentiviral vector (pfNL43-dE-EGFP) encoding HIV-1 (containing all nine viral genes: gag, pol, tat, rev, vif, vpr, vpu, nef, and env) was transfected into HEK293 cells (A) or electroporated to CD4+ Jurkat T lymphocytes (B), allowing reverse transcription and integration, followed by viral protein expression in cells. Neither the envelope protein nor any viral particles are produced because of the insertion of an EGFP cassette within the env gene. Expression levels of EGFP and two viral proteins, Rev and integrase, are shown in Fig S1A and B. This system allows in vitro simulation of HIV-1 infection in a safe, reproducible and quantifiable manner. Cells were lysed at indicated times post-transfection, and global sumoylation levels were assessed by Western blot, using human anti-SUMO1 and anti-SUMO2/3 antibodies. Representative blots are shown. Densitometric quantifications of SUMO signals were normalized to actin expression (which serves as a loading control). Data are presented as mean ± SEM (n = 4 for HEK293 cells, n = 7 for Jurkat cells), asterisks denote statistical significance (P-values were calculated using t test assuming unequal variances, ns, not significant). Control: cells transfected with an empty vector backbone devoid of HIV-1 genes, HIV, cells expressing the HIV-1 genome.