Deficiency in Either 4E-BP1 or 4E-BP2 Augments Innate Antiviral Immune Responses

<div><p>Genetic deletion of both 4E-BP1 and 4E-BP2 was found to protect cells against viral infections. Here we demonstrate that the individual loss of either 4E-BP1 or 4E-BP2 in mouse embryonic fibroblasts (MEFs) is sufficient to confer viral resistance. shRNA-mediated silencing of 4E-BP1 or 4E-BP2 renders MEFs resistant to viruses, and compared to wild type cells, MEFs knockout for either 4E-BP1 or 4E-BP2 exhibit enhanced translation of <i>Irf-7</i> and consequently increased innate immune response to viruses. Accordingly, the replication of vesicular stomatitis virus, encephalomyocarditis virus, influenza virus and Sindbis virus is markedly suppressed in these cells. Importantly, expression of either 4E-BP1 or 4E-BP2 in double knockout or respective single knockout cells diminishes their resistance to viral infection. Our data show that loss of 4E-BP1 or 4E-BP2 potentiates innate antiviral immunity. These results provide further evidence for translational control of innate immunity and support targeting translational effectors as an antiviral strategy.</p></div

Deficiency of 4E-BP1 or 4E-BP2 enhances type-I interferon production.

<p>(A) Protection assay: diagram of experimental protocol: after stimulation with poly(I:C) (6 hours) the medium of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were collected and transferred to three different plates containing WT MEFs. (B) WT MEFs from (A) were then infected with VSV-GFP. The protective effects of the different media were assessed 24 hpi by fluorescence microscopy, cytopathic effect and (C) virus titration. (D) WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were infected with VSV at a MOI of 1 PFU/cell for 6 hours and the induction of a type-I IFN response (<i>Irf-7</i>, <i>Ifn-α</i> and <i>Ifn</i>-β mRNA levels) was determined by RT-PCR (*longer exposure). (E) Luciferase assays showing the ratio of expression of the different luciferase constructs harbouring either 5′UTR-IRF-7 (translational level), IFN-α promoter or IFN-β promoter (transcriptional level), normalized to the transfection control (<i>Renilla</i> luciferase). Fluc activity/Rluc activity in WT MEFs was set as 1.</p

Silencing 4E-BP1 or 4E-BP2 inhibits VSV replication in MEFs.

<p>(A) Western blotting analysis of 4E-BP1 or 4E-BP2 expression following transduction of WT MEFs with lentiviruses containing a non-specific shRNA (scrambled), or a shRNA targeting <i>4E-BP1</i> or <i>4E-BP2</i> mRNA. β-actin served as a loading control. (B) Scrambled MEFs and MEFs knockdown on 4E-BP1 or 4E-BP2 were infected with VSV-GFP at a MOI of 1 PFU/cell and virus replication was assessed by GFP fluorescence and cytopathic effect (CPE). (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control. (D) Viral titer quantified by plaque assay at 24 hpi with VSV-GFP at a MOI of 1 PFU/cell.</p

Exogenous expression of 4E-BP1 or 4E-BP2 in respective 4E-BP1^−/− or 4E-BP2^−/− MEFs increases susceptibility to VSV infection.

<p>(A) 4E-BP1^−/− and 4E-BP2^−/− MEFs were transduced with retroviruses carrying an empty vector (control), or retroviruses expressing either 4E-BP1 or 4E-BP2. Western blotting analysis for exogenous expression of 4E-BP1 and 4E-BP2 in their respective single knockout MEFs. (B) Control and transduced MEFs were infected with VSV-GFP at a MOI of 1 PFU/cell and viral infection was assessed by GFP fluorescence and by (C) Western blotting analysis for VSV viral protein expression.</p

Exogenous expression of 4E-BP1 or 4E-BP2 in MEFs knockout for both translation repressors augments their susceptibility to VSV infection.

<p>(A) 4E-BP1/2 DKO MEFs were transduced with retroviruses expressing either 4E-BP1, 4E-BP2 or an empty vector as control. Western blotting analysis for the expression levels of exogenous 4E-BP1 or 4E-BP2 in DKO MEFs. (B) Control and 4E-BP1- or 4E-BP2-expressing DKO MEFs were infected with VSV-GFP at a MOI of 1 PFU/cell for 24 hours and infection was monitored by GFP fluorescence and CPE. (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control.</p

Lack of 4E-BP1 or 4E-BP2 renders MEFs refractory to VSV infection.

<p>(A) Western blotting analysis of 4E-BP1 or 4E-BP2 expression in WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs. β-actin served as a loading control. (B) WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs were mock-infected or infected at a MOI of 1 PFU per cell with VSV-GFP. Twenty-four hpi, viral infection was assessed by GFP fluorescence and CPE. (C) Western blotting analysis for the detection of VSV proteins at the defined time points post-infection of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with VSV-GFP at a MOI of 1 PFU/cell. β-actin was used as a loading control. (D) Viral titer quantified by plaque assay at 24 hpi in WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with VSV-GFP at a MOI of 1 PFU/cell. (E) Photomicrograph of CPE resulting from infections of WT, 4E-BP1^−/− and 4E-BP2^−/− MEFs with FLU, Sindbis and EMCV virus at 1MOI 12 hpi. (F) Cell viability in experiment in (E) was assessed by MTT assay.</p

Control of Translation and miRNA-Dependent Repression by a Novel Poly(A) Binding Protein, hnRNP-Q

<div><p>Translation control often operates via remodeling of messenger ribonucleoprotein particles. The poly(A) binding protein (PABP) simultaneously interacts with the 3′ poly(A) tail of the mRNA and the eukaryotic translation initiation factor 4G (eIF4G) to stimulate translation. PABP also promotes miRNA-dependent deadenylation and translational repression of target mRNAs. We demonstrate that isoform 2 of the mouse heterogeneous nuclear protein Q (hnRNP-Q2/SYNCRIP) binds poly(A) by default when PABP binding is inhibited. In addition, hnRNP-Q2 competes with PABP for binding to poly(A) in vitro. Depleting hnRNP-Q2 from translation extracts stimulates cap-dependent and IRES-mediated translation that is dependent on the PABP/poly(A) complex. Adding recombinant hnRNP-Q2 to the extracts inhibited translation in a poly(A) tail-dependent manner. The displacement of PABP from the poly(A) tail by hnRNP-Q2 impaired the association of eIF4E with the 5′ m⁷G cap structure of mRNA, resulting in the inhibition of 48S and 80S ribosome initiation complex formation. In mouse fibroblasts, silencing of hnRNP-Q2 stimulated translation. In addition, hnRNP-Q2 impeded let-7a miRNA-mediated deadenylation and repression of target mRNAs, which require PABP. Thus, by competing with PABP, hnRNP-Q2 plays important roles in the regulation of global translation and miRNA-mediated repression of specific mRNAs.</p></div

HnRNP-Q2 inhibits m⁷G cap structure recognition by translation initiation factors.

<p>(A–D) Inhibition of 80S and 48S initiation complex formation by hnRNP-Q2 in nuclease-treated RRL. 80S ribosome binding to 3′ end labeled globin mRNA was assayed in a cycloheximide (0.6 mM)-supplemented RRL, normal (A) or hnRNP-Q2-depleted (B), in the presence of control buffer (squares) or recombinant hnRNP-Q2 (15 µg/ml) (triangles). (C) Validation of the 48S pre-initiation complex formation in the presence of GMPPNP. GTP or GMPPNP were added to the reaction mixtures at 2 mM final concentration as indicated. Other conditions were similar to those described for panel B. (D) 48S pre-initiation complex formation in hnRNP-Q2-depleted RRL in the presence of GMPPNP and either control buffer (squares) or hnRNP-Q2 (25 µg/ml) (triangles). The reaction mixtures were analyzed on 5-ml 15%–30% (A and B) or 11-ml 10%–30% (C and D) sucrose gradients. (E) HnRNP-Q2 dose-dependent inhibition of eIF4E binding to the m⁷G cap structure in RRL as analyzed by chemical crosslinking. Control and hnRNP-Q2-depleted RRL were incubated with oxidized ³²P-cap-labeled poly(A)-extended Luc mRNA in the absence or presence of the indicated concentrations of recombinant hnRNP-Q2. The positions of eIF4E and eIF4A are indicated. Relative efficiencies of eIF4E crosslinking are indicated at the bottom (the value obtained for control RRL was set as 100%).</p

Competition between hnRNP-Q2 and PABP for binding to the poly(A) tail in RRL.

<p>(A) Western blot analysis of RRL depleted with either anti-FLAG (Control RRL) or anti-hnRNP-Q antibody. The blot was probed for anti-hnRNP-Q or anti-β-actin (loading control). (B) Proteins of control or hnRNP-Q2-depleted RRL that crosslink with the ³²P-poly(A) tail in the presence of the indicated concentrations of recombinant hnRNP-Q2. (C) Proteins of control or PABP-depleted RRL that crosslink to the ³²P-poly(A) tail in the presence of the indicated concentrations of recombinant PABP.</p

Poly(A) tail length and PABP-dependent inhibition of translation by hnRNP-Q2 in Krebs extract.

<p>(A) Krebs extract that was not nuclease-treated was programmed with Cap-Luc mRNAs (0.2 µg/ml) bearing poly(A) tails of the indicated length. Control buffer of hnRNP-Q2 (20 µg/ml) was added to the reaction mixtures as indicated. (B) Sequestering of PABP by Paip2 renders translation insensitive to the poly(A) tail length and inhibition by hnRNP-Q2. Cap-Luc mRNA with increasing poly(A) tails was translated in the untreated extract in the presence of Paip2 (15 µg/ml) as described for panel A. hnRNP-Q2 (20 µg/ml) was added to the reaction mixtures where indicated. Inhibition of translation by hnRNP-Q2 is shown on the top of the panels. (C) Endogenous [³⁵S]methionine incorporation in the untreated extract in the presence of the indicated concentrations of hnRNP-Q2 or 10 µM hippuristanol (Hipp). Incubation was at 32°C for 2 h. Average values for trichloroacetic acid-insoluble radioactivity in 1-µl aliquots of the samples from three assays with standard deviations are shown.</p