eIF4GI mediates the Ago2-cap association.

<p>(<i>A</i>) Identification of a translation initiation factor capable of augmenting the Ago2-cap association. WCEs from 293FT cells co-transfected with plasmids expressing myc-Ago2 and Flag-tagged translation factors (eIF3c, eIF4AI, eIF4E, eIF4GI and PABP) were subjected to cap-pulldown assays. The expression levels of the transfected genes (lanes 1–6), their cap-associations (lanes 7–12), and various proteins from WCEs or from the resin-bound fractions were monitored by Western blotting with the indicated antibodies. (<i>B</i>) The eIF4GI-dependent cap-association of Ago2. WCE from HeLa cells transfected with si-Control or si-eIF4GI were applied to cap-pulldown assays. The knock-down efficiency of siRNAs and the resin-bound proteins were monitored using antibodies described. (<i>C</i>) Determination of a domain in eIF4GI responsible for augmenting the Ago2-cap association. WCEs from 293FT cells transiently expressing myc-Ago2 and Flag-tagged fragments encoding the N-terminal, middle or C-terminal regions of eIF4GI (depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#pone-0055725-g005" target="_blank">Figure 5A</a>) were subjected to cap-pulldown assays. The resin-bound Ago2 (upper panel), the ectopically expressed Ago2 and eIF4GI fragments (lower panel), GAPDH and eIF4E proteins were detected using the indicated antibodies.</p

eIF4GI Facilitates the MicroRNA-Mediated Gene Silencing

<div><p>MicroRNAs (miRNAs) are small noncoding RNAs that mediate post-transcriptional gene silencing by binding to complementary target mRNAs and recruiting the miRNA-containing ribonucleoprotein complexes to the mRNAs. However, the molecular basis of this silencing is unclear. Here, we show that human Ago2 associates with the cap-binding protein complex and this association is mediated by human eIF4GI, a scaffold protein required for the translation initiation. Using a cap photo-crosslinking method, we show that Ago2 closely associates with the cap structure. Taken together, these data suggest that eIF4GI participates in the miRNA-mediated post-transcriptional gene silencing by promoting the association of Ago2 with the cap-binding complex.</p> </div

The intact Ago2 associates with the cap-binding complex.

<p>(<i>A</i>) A schematic diagram of human Ago2 and the constructs used for co-immunoprecipitation and cap-pulldown assays. (<i>B</i>) The domains in Ago2 required for the association with eIF4GI. 293FT cells were transfected with plasmids expressing Flag-tagged N-, M- or C-terminal portions of eIF4GI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#pone-0055725-g005" target="_blank">Figure 5A</a>) together with plasmids expressing myc-tagged Ago2 fragments corresponding to the N, PAZ/M or PIWI domains. Extracts from the transfected cells were incubated with the Flag-resin and the precipitated proteins were visualized by Western blotting using the indicated antibodies. (<i>C</i>) RNA-independent association of Ago2 domains with eIF4GI domains. RNase-treated (lanes 2, 4, 6, 8, 10 and 12) or -untreated (lanes 1, 3, 5, 7, 9 and 11) WCE from 293FT cells transiently expressing Flag-tagged eIF4GI derivatives (eIF4GI-N, -M or -C) with myc-tagged Ago2 variants (Ago2-N, -PAZ/M or -PIWI) were subjected to the immunoprecipitation experiments using the Flag-resins. The bound proteins were detected using antibodies indicated. (<i>D</i>) Association of Ago2 domains with the cap-resin. 2 mg of WCEs from 293FT cells transfected with plasmids expressing myc-tagged Ago2 derivatives were applied to cap-pulldown assays and the bound proteins were monitored using anti-myc antibodies. For comparison, various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5.</p

The 5′ cap structure may directly contact with Ago2.

<p>(<i>A</i>) WCEs (2 mg) from 293FT cells transfected with plasmids expressing Flag-Ago1, Flag-Ago2, Flag-Dicer, Flag-TNRC6C or Flag-eIF4E were subjected to Flag-IP, and cap-labeled RNAs were then incubated with the immunoprecipitated proteins in the presence or absence of cap analogs. The RNAs and proteins were photo-crosslinked by UV irradiation and then analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#s2" target="_blank">Materials and methods</a> (upper panel). In parallel, Western blot analyses with an anti-Flag antibody were used to measure the amounts of the precipitated Flag-tagged proteins (lower panel). The positions of the proteins on the gel are denoted by arrows. (<i>B</i>) The effect of eIF4GI on the cap-crosslinking of Ago2. The resin-bound proteins from Flag-IP experiments using 293FT cell extracts expressing Flag-Ago2 and myc-eIF4GI were subjected to a cap-crosslinking assay (upper panel) and Western blotting (lower panel). (<i>C</i>) The effect of the N-terminal part of eIF4GI on the cap-crosslinking of Ago2. 293FT cells were co-transfected with plasmids expressing Flag-Ago2 and myc-tagged eIF4GI-N (aa 42–622), and a cap-crosslinking assay was performed.</p

eIF4GI plays a role in the miRNA-mediated gene silencing of the poly(A)-tailed m⁷G-capped mRNA.

<p>(<i>A</i>) Schematic diagrams of the reporter mRNAs used here. The abbreviations of each reporter are depicted in parentheses at the left side. (<i>B</i>) The effect of the 5′ cap structure and the poly(A) tail on the miRNA-mediated translational repression. m⁷G-capped/A-capped and/or poly(A)-tailed/nonadenylated FL mRNAs were co-transfected with the nonadenylated m⁷G-capped RL mRNAs in the presence of miControl or miCXCR4 into HeLa cells. After 19 h of incubation, the luciferase activities were measured to obtain the FL/RL activity ratios. (<i>C</i>) Total RNAs from the cells in panel B were extracted, and the relative levels of the mRNAs were analyzed by quantitative RT-PCR analysis. In panels <i>B</i> and <i>C</i>, the values from miControl-treated cells in all experiments were represented as 100% (lane 1). (<i>D</i>) De-repression of the miRNA-mediated gene silencing by siRNAs against translation factors using the RNA reporter system. The overall procedures for experiments and analyses were described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055725#s2" target="_blank">Materials and Methods</a>. All experiments were performed in triplicate. In panels <i>B</i> and <i>D</i>, the P-values are described (Student <i>t</i>-test). Standard deviations are indicated by bars.</p

eIF4GI associates with Ago2.

<p>(<i>A</i>) Schematic diagram of human eIF4GI. ‘4E’ means ‘the eIF4E-binding motif’. Plasmids were constructed for expression of various Flag-tagged eIF4GI fragments in human cells. (<i>B</i>) The N-terminal and middle domains of eIF4GI participate in the eIF4GI-Ago2 association. Plasmids expressing Flag-tagged eIF4GI variants and myc-tagged full-length Ago2 were co-transfected in 293FT cells, and their associations were examined by Flag immunoprecipitation (Flag-IP) with the Flag-resin. The levels of Ago2 and the eIF4GI mutants (left panel) and the amount of co-precipitated Ago2 (right panel) were monitored by Western blotting using the indicated antibodies. (<i>C</i>) Schematic diagram of the N-terminal constructs of eIF4GI for fine mapping of the region required for the association with Ago2. (<i>D</i>) Determination of the Ago2-associated region in eIF4GI. WCEs from 293FT cells expressing myc-tagged full-length Ago2 and N-terminal variants of eIF4GI serially deleted from the C- or N-termini were subjected to Flag-IP. The expressions of Ago2 and the eIF4GI derivatives (lower panel) and the amount of precipitated Ago2 (upper panel) were examined using the indicated antibodies. (<i>E</i>) RNA-independent association of Ago2 with eIF4GI (aa 42–202). WCEs from 293FT cells expressing myc-tagged Ago2 and Flag-tagged eIF4GI-NtP were treated with (lanes 2 and 4) and without (lanes 1 and 3) RNase A and subjected to Flag-IP.</p

Human Ago associates with the cap-binding complex.

<p>(<i>A</i>) The cap-association of endogenous Ago2 proteins from HeLa cells were examined by a cap-pulldown assay, using 2 mg of whole-cell extracts (WCEs) for incubation with either control-resin (lane 6) or cap-resin in the presence (lane 8) or absence (lane 7) of the cap analog. (<i>B</i>) The cap-association of miRNAs. WCEs from HeLa cells (2 mg) were subjected to a cap-pulldown assay, and the cap-associated RNAs were extracted and subjected to UREA-PAGE followed by Northern blotting using radiolabeled probes against the indicated miRNAs. For comparison, various amounts equal to 4.4–0.8% of the total RNAs contained in WCEs used for the cap-pulldown assays (∼10–2 µg each) were loaded in lanes 1–5. (<i>C</i>) The cap-associations of ectopically expressed proteins were monitored as in panel <i>A</i>, except for using 2 mg of WCEs from 293FT cells transfected with plasmids expressing Flag-tagged Ago1, Ago2 or Dicer. (<i>D</i>) Cap-pulldown assays were done using 2 mg of WCEs from 293FT cells expressing Flag-Ago2 with 200 µM of G(5′)ppp(3′)G (lane 3) or m⁷G(5′)ppp(3′)G (lane 4). (<i>E</i>) The RNA-independent cap-association of Ago2. 2 mg of WCEs from 293FT cells ectopically expressing myc-tagged Ago2 were treated with (lanes 2 and 5) or without (lanes 1, 3 and 4) RNase A and subjected to cap-pulldown assays. In panels <i>A</i> and <i>C</i>, various amounts corresponding to 2–0.4% of WCEs used in the pulldown assay were loaded in lanes 1–5 for comparison. In panels <i>A</i>, <i>C</i>, <i>D</i> and <i>E</i>, Western blot analyses were performed using the indicated antibodies.</p

Empirical validation of prediction performance of the integrated approach.

<p>(A) Knockdown effects of candidate proteins in HCV replicon. RLuc-replicon cells were transfected with a negative control siRNA or siRNAs against the seven candidate genes. At 48 hrs after transfection, cells were harvested and lysed, and Renilla luciferase activity in the cell lysates was measured. Luciferase activities were normalized to the amounts of proteins in the cell lysates (mean ± s.d. from three independent experiments performed in duplicate). An siRNA against hnRNPD was used as a positive control. The relative luciferase activities in the lysates are depicted by setting the luciferase activity in the control lysate to 1. (B) Knockdown effects of candidate proteins on HCV infection. Huh7.5.1 cells were transfected with a negative control siRNA or siRNAs against the seven candidate genes. An siRNA against CLDN1 was used as a positive control. At 48 hrs after transfection of siRNAs, the cells were infected with JFH1 5A-Rluc virus (MOI of 0.3) and cultivated for an additional 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activity in cell extracts. The luciferase activities were normalized to the amounts of proteins in the cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in the lysates are depicted by setting the luciferase activity in the control lysate to 1. (C) The relative amounts of HCV RNA in the cells at 6 or 12 hrs after infection. Huh7.5.1 cells were transfected with a negative control siRNA, a positive control siRNA (CLDN1), or siRNAs against the seven candidate genes. At 48 hrs after the siRNA transfections, the cells were infected with JFH1 5A-Rluc virus (MOI of 5). At 6 or 12 hrs after HCV infection, cells were harvested, total RNAs were purified, and the amounts of HCV and GAPDH RNAs were measured by qRT-PCR. The amounts of HCV RNAs were normalized by those of GAPDH RNAs (mean ± s.d. from three independent experiments performed in duplicate). (D) Knockdown effects of candidate proteins on HCV entry. Huh7.5.1 cells were transfected with a negative control siRNA, a positive control siRNA (CLDN1), or siRNAs against the seven candidate genes. At 48 hrs after the siRNA transfections, the cells were infected with HCVpp or VSVGpp, and then cultivated for additional 48 hrs. Infectivities of HCVpp and VSVGpp were monitored by measuring Renilla luciferase activities in the cell extracts, and normalized to the amounts of proteins in the cell extracts. The relative luciferase activities in the lysates are depicted by setting the luciferase activity in the control lysate to 1 (mean ± s.d. from three independent experiments performed in duplicate).</p

Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

<div><p>Successful viral infection requires intimate communication between virus and host cell, a process that absolutely requires various host proteins. However, current efforts to discover novel host proteins as therapeutic targets for viral infection are difficult. Here, we developed an integrative-genomics approach to predict human genes involved in the early steps of hepatitis C virus (HCV) infection. By integrating HCV and human protein associations, co-expression data, and tight junction-tetraspanin web specific networks, we identified host proteins required for the early steps in HCV infection. Moreover, we validated the roles of newly identified proteins in HCV infection by knocking down their expression using small interfering RNAs. Specifically, a novel host factor CD63 was shown to directly interact with HCV E2 protein. We further demonstrated that an antibody against CD63 blocked HCV infection, indicating that CD63 may serve as a new therapeutic target for HCV-related diseases. The candidate gene list provides a source for identification of new therapeutic targets.</p> </div

Functional characterization of the top 100 ranked proteins predicted to be involved in the early steps of HCV infection.

<p>(A) Statistically significant overrepresentation of functional terms based on mapping of Gene Ontology (GO) biological process to the top 100 ranked human proteins (<i>P</i><1.0×10⁻² after correlation for Benjamini). (B) Significantly overrepresented KEGG signaling pathways (<i>P</i><1.0×10⁻² after correlation for Benjamini).</p