Fig 1.
Pimozide abolishes B19V replication in primary CD36+ erythroid progenitor cells (EPCs).
(A) Inhibition of phosphorylation of STAT5 by pimozide. CD36+ EPCs were treated with dimethyl sulfoxide (DMSO) vehicle or pimozide at various concentrations. At 48 h post-treatment, cells were collected, washed and lysed for Western blotting with detection of pSTAT5 by anti-pSTAT5(Y694) antibody. The blot was reprobed for total STAT5 using a mouse anti-STAT5 antibody and for β-actin as a loading control. (B-D) Inhibition of B19V DNA replication by pimozide. CD36+ EPCs were pre-incubated with DMSO or pimozide at a final concentration of 15 μM or 25 μM 6 h prior to B19V infection. At 48 h post-infection, cells were subjected to (B) flow cytometry analysis for the B19V-infected cell population with an anti-B19V capsid antibody, (C) Hirt DNA extraction, followed by Southern blot analysis with a B19V M20 DNA probe, or (D) Western blotting with an anti-pSTAT5(Y694) antibody, and reprobing with an anti-β-actin antibody. (E&F) Evaluation of the effect of pimozide on cell proliferation. CD36+ EPCs were treated with either DMSO or pimozide (15 μM or 25 μM). After 48 h, treated cells were incubated with bromodeoxyuridine (BrdU) for 1 h to analyze cell-cycle progression by a BrdU incorporation assay. (E) Results of a representative cell-cycle analysis experiment. (F) Relative fold change in the S phase cell population of each group is shown, with means and standard deviations of three independent experiments. P values are calculated using one-way and Tukey-Kramer post-test, compared with DMSO control. * denotes P<0.05; **** denotes P<0.0001; and n.s. (P>0.05) denotes no statistical significance.
Fig 2.
STAT5 interacts with B19V replication origins (Ori) in vitro.
(A) Diagram of the B19V ssDNA and replicative form (RF) DNA genome. B19V genomes of the single-stranded (ss) DNA form and full-length replicative form (RF) are depicted, along with the sequence of viral Ori that contains a consensus STAT5-binding element (STAT5BE), terminal resolution site (trs), two NS1-binding elements (NSBE1 and NSBE2), and two putative cellular factor-binding elements (CFBE) [38–40]. (B) Probes used in electrophoretic mobility shift assay (EMSA). Sequences of two 39-nt probes, wt-Ori-39 and mut-Ori-39, are shown with the consensus STAT5BE and the mutated STAT5BE (mSTAT5BE) highlighted. (C&D) EMSA. (C) 32P-labeled Ori probes wt-Ori-39 (lane 2) and mut-Ori-39 (lane 3) were incubated with UT7/Epo-S1 nuclear lysate (NL) in the presence of non-specific competitor poly dI-dC. Products were subjected to non-denaturing 5% polyacrylamide gel electrophoresis (PAGE). Gels were dried and exposed to a phosphor screen. (D) Similarly, EMSA was performed with 32P-labeled wt-Ori probes and 5 μg of NL in the presence of 5 μg of anti-pSTAT5(Y694) or IgG control antibody. (E) PAGE analysis of purified pSTAT5. 20 μl of pSTAT5 was analyzed by SDS-10% PAGE. Gels were either stained with Coomassie brilliant blue (left panel/CBB staining), or transferred to a PVDF membrane for Western blotting with an anti-pSTAT5(Y694) antibody (right panel/Western blot). (F&G) EMSA with purified pSTAT5. (F) 32P-labeled wt-Ori-39 (lane 2) and mut-Ori-39 (lane 3) probes were incubated with purified pSTAT5 in the presence of poly dI-dC. Samples were run on 5% non-denaturing PAGE, dried, and exposed to a phosphor screen. (G) EMSA with wt-Ori-39 in the absence (lanes 2&3) or presence of STAT5-SH2i at 0.3 mM (lane 4), 0.5 mM (lane 5), and 0.8 mM (lane 6). Lane 1, wt-Ori-39 probe only.
Fig 3.
STAT5 colocalizes with B19V NS1, capsids, and the replicating B19V genome.
(A&B) STAT5 colocalizes with B19V NS1 and capsids. Mock- or B19V-infected CD36+ EPCs were co-stained and examined with rabbit anti-STAT5 and rat anti-B19V NS1 antibodies (A) or with rabbit anti-STAT5 and mouse anti-B19V capsid antibodies (B). (C-E) Proximity ligation assay. Infected cells were co-stained with rabbit anti-STAT5 and mouse anti-B19V capsid antibodies (C), or co-stained with rabbit anti-STAT5 and mouse anti-BrdU antibodies (D), or co-stained with mouse anti-B19V capsid and rabbit anti-BrdU antibodies (E), followed by a proximity ligation assay, which produces amplified signal for labeled molecules in close proximity. (F) STAT5 colocalizes with the replicating viral genome. Mock- or B19V-infected CD36+ EPCs were BrdU labeled to identify replicating viral ssDNA genomes. The treated cells were co-stained with rabbit anti-STAT5 and mouse anti-BrdU antibodies, followed by incubation with secondary antibodies. Images were taken with an Eclipse C1 Plus (Nikon) confocal microscope at 100 × magnification. Nuclei were stained with DAPI.
Fig 4.
Chromatin immunoprecipitation (ChIP) assay.
ChIP assay was performed using either infected CD36+ EPCs or transfected UT7/Epo-S1 cells, as indicated. (A) Crosslinked chromatin was sheared by sonication to sizes of ~500 bp. (B) An anti-pSTAT5(Y694) antibody or negative control IgG was used to pull down DNA-protein complexes. Recovered DNA from UT7/Epo-S1 cells or CD36+ EPCs was examined for viral DNA by PCR with primer sets of F1/R1 and F1/R2, respectively, which span the Ori sequences of the B19V genome. pM20 plasmid was used as a template for positive controls of PCR. (C) A diagram of the Ori-targeting PCR. The primers used for PCR are shown.
Fig 5.
Blockage of interaction between STAT5 and B19V Ori DNA inhibits B19V replication.
(A) STAT5-SH2 inhibitor (STAT5-SH2i) abolishes the shift of viral Ori in EMSA. UT7/Epo-S1 nuclear lysate (NL) was incubated with 32P-labelled wt-Ori-39 probe (lanes 2–5) with the addition of DMSO (lane 3) or STAT5-SH2i at 0.4 mM (lane 4) and 0.8 mM (lane 5). (B-D) STAT5-SH2i significantly inhibits viral DNA replication. CD36+ EPCs were incubated with either DMSO or STAT5-SH2i (250 μM or 500 μM), 6 h prior to infection. At 48 h post-infection, cells were collected and subjected either to (B) flow cytometry analysis for the B19V-infected (B19V+) cell population, with an anti-capsid antibody, or to (C) Hirt DNA extraction for Southern blot analysis with a B19V M20 DNA probe (upper panel), with mitochondrial DNA (Mito DNA) probed as a loading control (lower panel), or to (D) protein extraction for Western blotting with anti-pSTAT5 and anti-β-actin (E&F) Effect of STAT5-SH2i on cell proliferation. CD36+ EPCs were treated with either DMSO or STAT5-SH2i (250 μM or 500 μM), and were then incubated with BrdU to perform BrdU incorporation assays. (E) Results of a representative cell-cycle analysis. (F) Relative fold changes in the S-phase cell population of each group shown with means and standard deviations of three independent experiments. P values are calculated using one-way ANOVA and Tukey-Kramer post-test (P>0.05), compared with DMSO control. **** denotes P<0.0001, ** P<0.01, and n.s. no statistical significance.
Fig 6.
Failure of B19V replicative form DNA clones with STAT5-binding element mutations in replication in transfected UT7/Epo-S1 cells.
(A) Diagram of the B19V full-length M20 RF genome and various half ITR-deleted N8 RF genomes with mutations at STAT5-binding elements (STAT5BEs). Red squares indicate the position of the Ori sequences at both ITRs, and grey squares indicate mutated Ori (mOri). The sequence of the mOri is shown with mutated nucleotides in grey in the STAT5BE. (B&C) Southern blot analysis. (B) The N8 RF DNA, or derivatives with mutations in the STAT5BE of either the left ITR (N8mOriL), right ITR (N8mOriR), or both (N8mOri), were transfected into UT7/Epo-S1 cells. (C) M20, and M20mOri, a derivative of the M20 RF DNA with STAT5BEs of both ITRs mutated, were transfected into UT7/Epo-S1 cells. At 48 h post-transfection, cells were collected for Hirt DNA extraction. And Hirt DNA samples were analyzed by Southern blotting with an M20 DNA probe. RF DNA (RF), ssDNA (ss), and Dpn I-digested DNA (shown with a line) are indicated. Mitochondrial DNA (Mito DNA) was used as a loading control (lower panels). (D) Viral protein expression of B19V DNA mutants. M20 or M20mOri transfected UT7/Epo-S1 cells were stained with anti-NS1 or anti-capsid antibodies. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.
Fig 7.
pSTAT5, but not NS1, interacts with the MCM complex.
(A) Immunoprecipitation (IP) assay. Cell lysates of NS1Flag-expressing UT7/Epo-S1 cells were prepared for pull-down assays with either anti-Flag-conjugated beads or control beads. Immunoprecipitated proteins were examined for the presence of MCM2 by Western blotting. Blots were reprobed with rabbit anti-pSTAT5(Y694), anti-E2F5, and anti-Flag antibodies. Detection of E2F5 was used as a positive control for NS1 IP. (B) Co-IP assay. UT7/Epo-S1 cells were collected, washed, and lysed with RIPA buffer. After centrifugation, the supernatant was incubated with either rabbit anti-pSTAT5(Y694) or control IgG antibody. Immunoprecipitated proteins were blotted for the presence of the MCM complex with an anti-MCM5 antibody and for pSTAT5 with rabbit anti-pSTAT5(Y694). (C) Reverse Co-IP assay. Reverse Co-IP was performed with an anti-MCM5 antibody. Immunoprecipitated proteins were examined for pSTAT5, MCM2, and MCM5, respectively. (D) Co-IP of lysates treated with DNase. UT7/Epo-S1 cell lysates, either treated or untreated with DNase (750 units of Benzonase) were incubated with anti-pSTAT5(Y694) or control IgG antibodies for Co-IP assay, and immunoprecipitated proteins were examined for MCM2 by Western blot analysis. (E-H) Immunofluorescence analysis. (E&F) Mock- or B19V-infected CD36+ EPCs were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies, followed by (E) incubation with respective secondary antibodies, or by (F) proximal ligation assay, which produces amplified signal for labeled molecules in close proximity. (G) CD36+ EPCs were incubated with either DMSO or pimozide (at 30 μM) for 2 days. And then the cells were co-stained with rabbit anti-STAT5 and mouse anti-MCM2 antibodies for proximity ligation assay. (H) Infected EPCs were stained with an anti-capsid antibody. Confocal images were taken with an Eclipse C1 Plus (Nikon) microscope at 100 × magnification.
Fig 8.
The MCM complex is loaded onto the viral Ori.
(A) Experimental strategy. The STAT5-MCM complex is depicted interacting with a DNA sequence, such as the viral Ori. The time line of B19V infection and treatment with the STAT5-SH2 inhibitor (STAT5-SH2i) shows time (h) post-infection or post-treatment. (B) ChIP assay. Cells were treated as shown in panel A. An anti-MCM2 antibody was used to pull down the STAT5-MCM-DNA complex, and the recovered ChIP DNA was subjected to qPCR with a primer set spanning the Ori region. Compared with the absence of the STAT5-SH2i, the relative abundance (percentage) of MCM on the viral Ori in the presence of the STAT5-SH2i is shown, with mean and standard deviation of three independent experiments. P values are calculated using a Student’s t test, ** denotes P<0.01. (C) Three color confocal microscopy. Mock- or B19V-infected CD36+ EPCs were co-stained with rabbit anti-STAT5, rat anti-NS1, and mouse anti-MCM2 antibodies, followed by staining with secondary antibodies conjugated with Dylight405, Texas Red, and FITC, respectively. Images were taken with an Eclipse C1 Plus (Nikon) confocal microscope at 100 × magnification.
Fig 9.
IC50 determination and colony formation assay.
(A) IC50 determination. CD36+ EPCs incubated with pimozide at different concentrations were infected with B19V. After 48 h post-infection, the cells were extracted for Hirt DNA. The DNA samples were examined for B19V DNA with Southern blot analysis using a M20 RF DNA probe. Viral RF DNA was quantified. RF DNA levels with pimozide relative to levels without pimozide are plotted against the concentrations of pimozide for the calculation of the IC50 value with GraphPad Prism. (B&C) Colony formation assay. CD36+ EPCs were incubated with pimozide at different concentrations for 48 h, and then cultured in methyl cellulose-containing medium. After 10 days, numbers of the colonies were counted (B). P values are calculated using one-way ANOVA followed by Tukey-Kramer post-test, compared with DMSO group. ** P<0.01; * P< 0.05; n.s. (P>0.05) denotes no statistical significance. Images of colonies were taken with Eclipse C1 Plus (Nikon) inverted microscope at 10 × and 40 × magnifications (C).