Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex
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.