Fig 1.
PCV2 DNA interacts with SUMOylated host protein NPM1.
(A) Schematic diagram of the experiment design. The biotin-labeled PCV2 DNA was transfected into PK-15 cells for 24 h, and then the cells were infected with 1 MOI PCV2 or mock for 12 h. The PCV2 DNA binding host proteins were pulled down using streptavidin-conjugated beads. The PCV2 DNA pulled-down proteins were further immunoprecipitated with an anti-Pan SUMO antibody, and the precipitated proteins were analyzed by Liquid Chromatography-Mass Spectrometer/Mass Spectrometry (LC-MS/MS). (B) The PCV2 DNA pulled-down proteins were detected for the SUMOylation by western blot. (C) The SUMOylated proteins interacting with PCV2 DNA were analyzed using KEGG enrichment and GO enrichment analysis on KOBAS (http://kobas.cbi.pku.edu.cn/). Each row represents an enriched function, and the length of the bar represents the enrichment ratio. The color of the bar represents different clusters. For each cluster, if there are more than 5 terms, the top 5 with the highest enrich ratio are displayed. (D) The PCV2 DNA pulled-down proteins were detected using anti-NPM1 antibodies by western blot. (E) The anti-NPM1 antibody detected protein was analyzed by LC-MS/MS. One of the typical peptide fragments (251 aa-273 aa) was shown. (F) PK-15 cells were infected with 1 MOI PCV2 for 12 h, and then the NPM1 interacting DNA was analyzed by ChIP assays using PCV2 DNA-specific primers. (G) PK-15 cells were pre-treated with the SUMOylation inhibitors 2-D08 (200 μM) or ML-792 (10 μM) for 24 h, and then the cells were infected with 1 MOI PCV2 for 12 h with the presence of 2-D08 or ML-792. The NPM1 interacting DNA was analyzed by ChIP assays using PCV2 DNA-specific primers.
Fig 2.
The K263 SUMOylation site on the C-terminal domain of porcine NPM1 is critical for the interaction of porcine NPM1 and PCV2 DNA.
(A) Schematic diagram of the structure of human NPM1 (hNPM1) and porcine NPM1 (pNPM1), as well as the truncates of pNPM1. The red lines indicate the sites of the different amino acids between hNPM1 and pNPM1. The green arrows indicate the K230 and K263 SUMOylation sites on pNPM1. (B, C) Equal amounts of plasmids expressing the GFP-fused pNPM1 truncates were transfected into the pNPM1 knockout PK-15 (PK-15npm1-/-) cells for 24 h, then the cells were infected with 1 MOI PCV2 for 12 h. (B) The interaction of the pNPM1 truncates with PCV2 DNA was measured by ChIP assays. (C) The interaction of the pNPM1 truncates with PCV2 Cap was measured by co-IP assays. (D) The potential SUMOylation sites on pNPM1 were predicted by four online software, including SUMOplot (https://www.abcepta.com/sumoplot), JASSA (http://www.jassa.fr/), GPS-SUMO (http://sumosp.biocuckoo.org/), and Ron Hay (https://www.lifesci.dundee.ac.uk/groups/ron_hay/pages/SumomotifQuery.html). Details of the SUMO conjugation motifs and a summary of prediction software scores are depicted in the table. S = strict, R = relaxed, and RR = relaxed reverted in Ron Hay analysis. The short horizontal lines in the table represent have not been predicted by the analysis. (E) The same amount of the SUMOylation sites mutant pNPM1 expressing plasmids (K230R, K263R, and K230/263R) were transfected into PK-15npm1-/- cells for 24 h, and then the cells were infected with 1 MOI PCV2 for 12 h. The interactions of the SUMOylation sites mutated pNPM1 with PCV2 DNA were measured by ChIP assays. (F) The plasmids expressing Flag-tag labeled SUMOylation sites mutated pNPM1 as well as the GFP-tag labeled PCV2 Cap were transfected into PK-15npm1-/- cells for 24 h, and then the interaction of the SUMOylation sites mutated pNPM1 with PCV2 Cap were measured by co-IP assays.
Fig 3.
The SUMOylation facilitates the nucleoli localization of pNPM1 that promotes PCV2 DNA replication.
PK-15npm1-/- cells were transfected with equal amounts of the wild-type pNPM1, pNPM1 (K230R), pNPM1 (K263R), or pNPM1 (K230/263R) for 24 h, respectively. Then the cells were infected with 1 MOI PCV2 for 12 h. (A) The localizations of the wild-type pNPM1 (WT), pNPM1 (K230R), pNPM1 (K263R), or pNPM1 (K230/263R) were measured using confocal microscopy. The bars = 10 μm were indicated in each panel. (B) The cells were harvested and lysed with a nuclear and cytoplasmic protein extraction kit, then the expression of the pNPM1 and Cap were measured by western blot. (C) The PCV2 TCID50 of these cells were measured and calculated. (D) The copy numbers of PCV2 DNA were detected by qPCR and calculated. (E, F, G) The PCV2 genomes (CP) or replication form DNA (RFP) were detected using fluorescently-labeled specific DNA probes. (E) The positive cells were photographed by a fluorescence microscope. The bars = 100 μm were indicated in each panel. (F) The percentages of CP-positive cells were quantified per 500 cells. (G) The percentages of RFP-positive cells were quantified per 500 cells. **p < 0.01 versus the wild-type pNPM1 transfected cells in (C), (D), (F), and (G).
Fig 4.
PCV2 infection promotes SUMO2/3 binding to pNPM1 within 24 h.
(A) PK-15 cells were infected with 1 MOI PCV2 or mock (the same volume of medium) for 0 h, 6 h, 12 h, 18 h, and 24 h. The expression levels of pNPM1, SUMOylated pNPM1, and PCV2 Cap were detected by western blot. The expression levels of pNPM1 and SUMOylated pNPM1 relative to β-actin were calculated. (B) Equal amounts of plasmids expressing the pNPM1 SUMOylation site mutants were transfected into PK-15npm1-/- cells for 24 h, and then the cells were infected with PCV2 for 12 h. The expression levels of pNPM1 and SUMOylated pNPM1 were detected by western blot and calculated. (C, D, E, F) The PK-15npm1-/- cells were transfected with Flag-tagged pNPM1 and GFP-fused SUMO proteins, respectively. Then the cells were infected with mock or 1 MOI PCV2 for 12 h. (C) The localizations of pNPM1 and SUMO proteins in these cells were detected by confocal microscopy. The bars were indicated in each panel. The interactions of pNPM1 and SUMO1 (D), SUMO2 (E), or SUMO3 (F) were measured by IP assays, the pEGFP-N1 represents the vector control of SUMO proteins. **p < 0.01 versus the mock-infected cells in (A), versus the wild-type pNPM1 transfected cells in (B). ## p < 0.01 versus the PCV2-infected cells at 18 h post-infection in (A). ND represents not detected.
Fig 5.
PCV2 infection promotes Ubc9/TRIM24 signalings to enhance the SUMOylation of pNPM1.
(A, B) 1 MOI PCV2 or mock (the same volume of medium) infected PK-15 cells for 0 h, 6 h, 12 h, 18 h, and 24 h. (A) The expression levels of SAE1, Ubc9, TRIM24, TRIM62, and SENP3 were analyzed by western blot. (B) The expression levels of Ubc9 and SENP3 relative to β-actin were calculated. (C) The plasmids expressing TRIM24, TRIM62, and pNPM1 were transfected into 293T cells for 48 h, and the interactions of TRIM24 and TRIM62 with pNPM1 were detected by co-IP assays. (D) The plasmids expressing TRIM24, TRIM62, and pNPM1 were transfected into PK-15npm1-/- cells for 24 h, and then the cells were infected with 1 MOI PCV2 or mock for 12 h. The interactions of TRIM24 and TRIM62 with pNPM1 were detected by co-IP assays. (E) The specific siRNAs targeting TRIM24 or TRIM62 were transfected into PK-15 cells for 24 h, then the cells were infected with 1 MOI PCV2 for 12 h. The expression levels of TRIM24, TRIM62, pNPM1, and SUMOylated pNPM1 were detected by western blot and calculated. (F) The specific siRNA targeting Ubc9 was transfected into PK-15 cells for 24 h, then the cells were infected with 1 MOI PCV2 for 12 h. The interactions of TRIM24 with pNPM1 were detected by co-IP assays. **p < 0.01, *p < 0.05 versus the mock-infected cells at the same time point in (B), **p < 0.01 versus the siNC-transfected cells in (E), ##p < 0.01 versus the PCV2-infected cells at 18 h in (B).
Fig 6.
PCV2 Cap is the major component promoting pNPM1 SUMOylation with the activation of the ERK pathway.
(A) PK-15 cells were infected with 100 MOI of the blank recombinant adenovirus (rAd-blank), the recombinant adenovirus expressing PCV2 Rep (rAd-Rep), the recombinant adenovirus expressing PCV2 Cap (rAd-Cap), or the recombinant adenovirus expressing PCV2 ORF3 (rAd-ORF3) for 24 h. The expressions of pNPM1, Rep, Cap, and ORF3 were analyzed by western blot. The expression levels of pNPM1 were calculated. (B) PK-15 cells were infected with 100 MOI rAd-blank or rAd-Cap for 24 h. The expression levels of Ubc9 and TRIM24 were analyzed by western blot and calculated. (C) PK-15 cells were infected with 100 MOI rAd-blank or rAd-Cap for 24 h. The interactions of TRIM24 with pNPM1 were detected by co-IP assays. (D) PK-15 cells were pre-treated with Control, DMSO, or specific inhibitors for PI3K/Akt (LY294002, 10 μM), ERK (PD98059, 20 μM), p38 (SB203580, 10 μM), and JNK (SP6000125, 10 μM) for 2 h, respectively. Then the cells were infected with 1 MOI PCV2 for 12 h with the presence of the inhibitors. The expressions of pNPM1 were analyzed by western blot, and the expression levels of pNPM1 and SUMOylated pNPM1 relative to β-actin were calculated. (E) PK-15 cells were pre-treated with DMSO or PD98059 for 2 h, and then infected with 100 MOI rAd-Cap for 24 h with the presence of PD98059. The expressions of pNPM1 were analyzed by western blot, and the expression levels of pNPM1 and SUMOylated pNPM1 relative to β-actin were calculated. (F, G) PK-15 cells were pretreated with DMSO or PD98059 for 2 h. The cells were infected with 1 MOI PCV2 for 12 h with the presence of PD98059. (F) The expression levels of Ubc9 were analyzed by western blot and calculated. (G) The interaction of TRIM24 with pNPM1 were detected by co-IP assays. **p < 0.01 versus the rAd-blank-infected cells in (A) and (B), **p < 0.01 versus the DMSO-treated cells in (D), (E), and (F).
Fig 7.
The ERK-specific siRNA treatment suppresses the nucleolar localization of pNPM1 and the replication of PCV2 DNA.
(A) PK-15 cells were transfected with the siRNA specific for ERK (siERK) or negative control siRNA (siNC) for 24 h and then infected with 100 MOI rAd-Cap or rAd-blank for another 24 h. The localizations of pNPM1 in these cells were measured by confocal microscopy. The bars = 10 μm were indicated in each panel. (B) The cells were harvested and lysed with a nuclear and cytoplasmic protein extraction kit, then the expression of the pNPM1 and Cap were measured by western blot. (C-J) PK-15 cells were transfected with the specific siERK or siNC for 24 h and then infected with 1 MOI PCV2 for 12 h. (C) The localizations of pNPM1 in these cells were measured by confocal microscopy. The bars = 10 μm were indicated in each panel. (D) The cells were harvested and lysed with a nuclear and cytoplasmic protein extraction kit, then the expression of the pNPM1 and Cap were measured by western blot. (E) The interactions of the pNPM1 with PCV2 DNA were measured by ChIP assays. (F) The TCID50 of PCV2 in these cells were measured and calculated. (G) The copy numbers of PCV2 DNA were detected by qPCR and calculated. (H, I, J) The PCV2 genomes (CP) or replication form DNA (RFP) of the PCV2-infected siERK-transfected cells were detected using fluorescently-labeled specific DNA probes. (H) The positive cells were photographed by a fluorescence microscope. The bars = 100 μm were indicated in each panel. (I) The percentages of CP-positive cells were quantified per 500 cells. (J) The percentages of RFP-positive cells were quantified per 500 cells. *p < 0.05, **p < 0.01 versus the siNC transfected cells in (E), (F), (G), (I) and (J).