Ago2 and HuR associate with the EV71 RNA in infected cells.

<p><b>(A)</b> Lysates of mock- or EV71-infected SF268 cells were prepared and analyed by ribonucleoprotein immunoprecipitation (RIP) with non-immune serum (N.I.) or Ago2 or HuR anti-sera. EV71 RNA was analyzed by Northern blotting. <b>(B)</b> Portions of immunoprecipitated materials were analyzed by Western blot to verify anti-Ago2 or anti-HuR–dependent recovery of Ago2 or HuR, respectively.</p

Effect of AUF1 on EV71 IRES activity and viral replication.

<p>(<b>A</b>) SF268 cells were transfected with plasmids expressing shAUF1 or shCTRL. After 2 days, extracts were prepared for Western blot analyses of AUF1 and β-actin (loading control). A 2-fold dilution series of extract from cells expressing shCTRL (lanes 1 to 4) permitted estimates of AUF1 knockdown efficiency (lane 5). (<b>B</b>) Effect of AUF1 knockdown on EV71 IRES activity. The diagram depicts the bicistronic reporter plasmid used to synthesize RNA for transfections and dual-luciferase assays. A control plasmid contained the IRES in the antisense (AS) orientation. SF268 cells were transfected with plasmids expressing shCTRL, shAUF1, or no shRNA (untrans.). Two days after transfection, RLuc-EV71-5′UTR-FLuc RNA was transfected into the cells. Luciferase activity was measured 2 days later. Mean values and standard deviations from three independent experiments are shown in the bar graph. (<b>C</b>) Schematic diagram of the IRES-dependent, monocistronic reporter for RNA synthesis. Monocistronic RNA containing the EV71 IRES and FLuc was transfected into cells expressing shCTRL, shAUF1, or no shRNA (untrans.). FLuc activity was measured as described in panel (B). Mean values and standard deviations from three independent experiments are shown in the bar graph. (<b>D</b>) Schematic diagram of the cap-dependent, monocistronic reporter. SF268 cells expressing shCTRL, shAUF1, or no shRNA (untrans.) were transfected with cap-dependent reporter RNA. RLuc activity in cell lysates was analyzed two days later. Mean values and standard deviations from three independent experiments are shown in the bar graph. (<b>E</b>) Effect of AUF1 knockdown on EV71 RNA levels. Cells were transfected with plasmids expressing shCTRL or shAUF1 or were left untransfected (untrans.). Two days later, cells were infected with EV71 at an MOI of 40 for 8 h. Total RNA was extracted and viral positive-strand RNA levels were determined by qRT-PCR. Mean values and standard deviations from three independent experiments are shown. (<b>F</b>) Effect of AUF1 knockdown on EV71 3C protease levels. SF268 cells were transfected with plasmids expressing shAUF1 or shCTRL. Cells were mock infected or infected with EV71 2 days after transfection. Cell lysates were analyzed by Western blotting with anti-3C antibody. Actin served as a loading control. (<b>G</b>) Replication of EV71 in AUF1-depleted cells. SF268 cells expressing shAUF1 or shCTRL were infected with EV71 at an MOI of 40 and incubated at 37°C. Medium was harvested 4 and 8 h post-infection (p.i.) and assayed for infectious virus by plaque formation with Vero cells. Mean values and standard deviations from three independent experiments are shown.</p

Ago2 and HuR associate with SL-II within the EV71 5’UTR.

<p><b>(A)</b> Secondary structures within the 5’UTR were predicted by MFold as depicted earlier [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140291#pone.0140291.ref015" target="_blank">15</a>]. Numbers below each stem-loop indicate 5’UTR nucleotides corresponding to each stem-loop. The RNA substrates used for protein-RNA pull-down experiments also contained the flanking region immediately 5’ to a given stem-loop. <b>(B)</b> Identification of Ago2 and HuR interaction sites within the EV71 5’UTR. RNA-protein pull-down experiments were performed to examine the interaction between Ago2, HuR, and AUF1 with the EV71 5’UTR segments. Biotinylated RNAs corresponding to the indicated stem-loops and immediate 5’ flanking region were synthesized; control RNAs lacked biotin. Recovered proteins in the pull-downs were detected by Western blotting analyses. As expected, AUF1 binds SL-II [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140291#pone.0140291.ref015" target="_blank">15</a>].</p

EV71 infection leads to cytoplasmic accumulation of AUF1.

<p>RD cells were mock infected or infected with EV71 at an MOI of 40. At 4, 6, and 8-conjugated, goat anti-rabbit IgG or tetramethyl rhodamine isothiocyanate-conjugated, goat anti-rat IgG was used as a secondary antibody. DAPI was used to stain nuclei. Images were captured by confocal laser scanning microscopy.</p

vsRNA1 enhances association of AUF1, Ago2, and HuR with SL-II.

<p>The pull-down assay was performed with SF268 lysate and biotin-labeled SL-II as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140291#pone.0140291.g002" target="_blank">Fig 2</a>. Additionally, the indicated pmoles of synthetic mimic vsRNA1 or synthetic scrambled RNA were added to reaction mixtures to examine the effects on AUF1, Ago2, and HuR binding to biotin-labeled SL-II RNA. Eluted proteins were analyzed by Western blot using antibodies to the indicated proteins.</p

AUF1 interacts with the EV71 5′UTR.

<p>(<b>A</b>) RNA-protein pull-down experiments were performed to examine the interaction between AUF1 and the EV71 5′UTR. The EV71 5′UTR was synthesized in vitro in the presence of biotin-UTP. Biotinylated RNA was incubated with SF268 cell lysate (200 µg proteins) for 15 min at 30°C. Unlabeled RNA was added to parallel reactions as a negative control. Streptavidin-linked beads were used to pull-down biotin-labeled EV71 5′UTR and its associated cellular proteins. The beads were washed and then resuspended in SDS-PAGE loading buffer to dissociate proteins from RNA. Samples were boiled and analyzed by Western blot using anti-AUF1 antibody. (<b>B</b>) Secondary structures within the 5′UTR were predicted by MFold. Numbers below each stem-loop indicate 5′UTR nucleotides encompassing the respective stem-loop. RNA substrates used for protein-RNA pull-down experiments contained the indicated stem-loop and the flanking region immediately 5′ to it. (<b>C</b>) Identification of AUF1 interaction site(s) within the EV71 5′UTR. Biotinylated RNAs containing the indicated stem-loops were synthesized; control RNAs lacked biotin. SL-VI extends to nt 745, so it contains linker sequence between the IRES and the AUG. RNA-protein pull-downs and Western blot analysis were carried out as described for panel (A). The blot was stripped and re-probed with anti-FBP1 antibody as a positive control for RNA-binding activity.</p

Effect of depleting AUF1 and/or hnRNP A1/A2 on EV71 IRES activity.

<p>SF268 cells were transfected with the indicated siRNAs and/or shRNA plasmids for two days. RLuc-EV71-5′UTR-FLuc RNA was then transfected into cells. Levels of hnRNP A1/A2 and AUF1 were analyzed by Western blot and luciferase activity was measured 2 days later. Mean values and standard deviations from three independent experiments are shown in the bar graph. Each lane of the Western blot corresponds to the siRNA/shRNA transfections indicated at the bottom of the bar graph.</p

AUF1 associates with EV71 RNA in infected cells.

<p>(<b>A</b>) Lysates of mock- or EV71-infected SF268 cells were prepared and analyzed by ribonucleoprotein immunoprecipitation (RIP) with non-immune serum or AUF1 anti-serum. Both input and immunoprecipitated materials were analyzed by Northern blotting for EV71 RNA. NI, RIP with non-immune serum. (<b>B</b>) Aliquots of immunoprecipitated materials were analyzed by Western blot to verify anti-AUF1–dependent recovery of AUF1. The AUF1 isoforms are indicated.</p

Association of AUF1, Ago2, and HuR with SL-II is independent of each other.

<p><b>(A)</b> Left panels: Levels of AUF1, Ago2 and HuR were reduced individually by transfection of SF268 cells with plasmids expressing the indicated shRNA, or shCTRL as a control. Knockdown efficiencies were estimated by comparisons of Western blots of lysates from cells expressing target shRNAs with serial dilutions of lysate from cells expressing shCTRL. Actin served as a loading control. Right panel: The pull-down assay was carried out as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140291#pone.0140291.g002" target="_blank">Fig 2</a>. Recovered proteins were analyzed by Western blot using the antibodies against the indicated proteins (right panel). <b>(B)</b> Top panels: Levels of AUF1, Ago2 and HuR were reduced in combinations of two proteins by transfection of SF268 cells with plasmids expressing the indicated shRNAs, or shCTRL as a control. Knockdown efficiencies were estimated by comparisons of Western blots of lysates from cells expressing target shRNAs versus lysate from cells expressing shCTRL. Actin served as a loading control. Bottom panel: Proteins recovered from individual pull-down assays were analyzed by Western blot using the antibodies against the indicated proteins.</p

AUF1 can compete with hnRNP A1 for association with the IRES.

<p>(<b>A</b>) The pull-down assay was performed with SF268 lysate and biotin-labeled EV71 5′UTR as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103827#pone-0103827-g002" target="_blank">Figure 2</a>. Additionally, increasing amounts of purified recombinant p40^AUF1 were added to reaction mixtures to examine the effects on hnRNP A1 binding to biotin-labeled EV71 5′UTR. Eluted proteins were analyzed by Western blot using anti-hnRNP A1 antibody (upper panel) or AUF1 antibody (lower panel). Lane 1: non-biotinylated RNA was added to the reaction as a negative control. Lanes 3–6: 0.2, 0.5, 1, and 1.5 µg of purified p40^AUF1 was added to reactions, respectively. (<b>B</b>) The pull-down assay was performed as described in panel (A) except increasing amounts of purified recombinant FBP1 were added to the reaction mixtures. (<b>C</b>) Biotin-labeled SL-II was used in the pull-down assay to examine the effect of increasing amounts of p40^AUF1 on hnRNP A1–SL-II interactions. (<b>D</b>) Biotin-labeled SL-II and increasing amounts of FBP1 were used in the pull-down assay to assess the effect of FBP1 on hnRNP A1–SL-II interactions.</p