Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

<div><p>The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES <i>trans</i>-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP1^1-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP1^1-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP1^1-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.</p></div

<i>In vitro</i> induction of FBP1 cleavage by EV71 viral proteinase 2A.

<p>(A) 10 μg of wild-type 2A^pro (2A) or mutant 2A^pro (2A^C110S), wild-type 3C^pro (3C) or mutant 3C^pro (3C^C147S) viral proteinases were added to RD cell lysates and incubated for 4 hours at 37°C. Cleavage of eIF4G and CstF-64 respectively served as positive controls for 2A^pro and 3C^pro activity. (B) [³⁵S] methionine-labeled FBP1 was incubated with purified EV71 2A^pro or mutant 2A^pro (2A^C110S) for 4 hours at varying doses, or for (C)15 minutes to 4 hours at a fixed dose of 5 μg at 37°C. Proteins were then separated by SDS-PAGE and analyzed by autoradiography. Cp-N and Cp-C: Cleavage products of FBP1.</p

Function, expression and subcellular localization of FBP1 in EV71-infected cells.

<p>(A) RD cells were transduced with lentivirus carrying shNC (control) or shFBP1. Knockdown efficiency of FBP1 was confirmed by immunoblotting with antibodies that recognize the N-terminal epitope (Ab-N) of FBP1. Signal intensities of immunoblotting were quantified by ImageJ software, and the ratios of FBP1 intensity to actin are shown at bottom. <i>In vitro</i> (B) IRES-dependent and (C) cap-dependent translation were performed using EV71 5′ UTR-FLuc monocistronic RNA. Reactions were incubated with shNC or shFBP1 RD cytoplasmic extracts in the presence or absence of 250 nM of recombinant FBP1, and (D) 50% of the translation reactant were subjected to immunoblotting. Luciferase activity exhibited by the reporter was monitored with a luminometer. Error bars represent the standard deviation from three experimental and three technical replicates. P value was determined by two-tailed Student’s t test (**, <i>p</i> < 0.01). (E) Proteins expressed by RD cells at 2–10 hours post-infection (h.p.i.) by EV71 were analyzed by immunoblot analysis using anti-FBP1, anti-3D^pol, and anti-actin antibodies. Cp: Cleavage product of FBP1. Viral RNA was extracted at 2–10 h.p.i. and analyzed by slot-blotting. (F) Proteins in the cytoplasmic and nuclear fractions taken from cells at 2–6 h.p.i. were analyzed by immunoblot analysis, using anti-FBP1, anti-Lamin A/C, anti-GAPDH, and anti-3D^pol antibodies. M: mock-infected cells; C: cytoplasmic fraction; N: nuclear fraction.</p

FBP1 cleavage following EV71 infection is independent of cellular proteasome, lysosome, or caspase activities.

<p>(A) EV71-infected or mock-infected RD cells were treated with 20 μM MG132 or 20 mM NH₄Cl at 3 h.p.i. Cell extracts at the indicated time points were analyzed with anti-FBP1, anti-3D^pol and anti-actin antibodies. Viral 3D^pol was used as an indicator for viral replication. (B) Cells were treated with 20 μM QVD-OPh, and lysates from the indicated time points were analysed with anti-FBP1, anti-PARP, anti-3C and anti-actin antibodies. Detection of PARP and its cleavage product (PARP Cp) was used as a positive control for viral-induced caspase activity. Viral 3C protein was used as an indicator for virus infection, and actin served as a loading control. Cps: Cleavage products of FBP1.</p

Additive effects of FBP1 and FBP1^1-371 on IRES-driven translation.

<p>(A) FBP1 and FBP1^1-371 impact on EV71 IRES activity <i>in vitro</i>. EV71 5′ UTR-FLuc RNA was translated with shFBP1-RD cytoplasmic extracts, in the presence of increasing amounts of recombinant FBP1 or FBP1^1-371. Reactions without FBP1 or FBP1^1-371 were used as controls. Luciferase activity exhibited by the reporter was monitored with a luminometer. (B) Additive effect of FBP1 and FBP1^1-371 on EV71 IRES-driven translation <i>in vitro</i>. Recombinant FBP1, FBP1^1-371 or a combination of FBP1 and FBP1^1-371, were respectively added with EV71 5′ UTR-FLuc RNA to shFBP1-RD cytoplasmic extracts. A reaction without FBP1 or FBP1^1-371 was used as a control. (C) Effects of FBP1 and FBP1^1-371 binding to the linker region on EV71 IRES-driven translation <i>in vitro</i>. Recombinant FBP1, FBP1^1-371 or a combination of FBP1 and FBP1^1-371, were respectively added to wild-type EV71 5′ IRES-FLuc RNA, FBP1^1-371 binding site mutant EV71-IRES-mB1-FLuc RNA, FBP1 binding site mutant EV71-IRES-mB2-FLuc RNA, or FBP1 and FBP1^1-371 binding site-double mutant EV71-IRES-mB1B2-FLuc RNA in shFBP1-RD cytoplasmic extracts. A reaction without FBP1 or FBP1^1-371 was used as a control. (D) EV71 replicon 3D^D330A (upper panel), which is defective in viral RNA replication, was used to determine the effects of FBP1 and FBP1(G371K) on viral protein translation. shFBP1-RD cells were transiently transfected with a vector control or plasmids expressing FLAG-tagged human FBP1 or FBP1(G371K) wobble mutant [FBP1^R and FBP1(G371K)^R], which are resistant to the targeting of shFBP1. At 48 hours post-transfection, the cells were subsequently transfected with EV71 replicon 3D^D330A RNA. Luciferase activity exhibited by the cells were then monitored using a luminometer at 6 hours after the second transfection. Relative amounts of transfected replicon RNA and protein expression levels in each experimental set were also tested. The experiments conducted in A-D were repeated three times, and each sample was prepared in triplicate, while the results were analyzed statistically by Student’s t-test. Error bar: standard deviation; *: <i>p</i> <0.05 and **: <i>p</i> <0.01. (E) shFBP1-RD cells were transiently transfected with a vector control or plasmids expressing FLAG-tagged FBP1^R or FBP1(G371K)^R. At 48 hours post-transfection, the cells were subsequently infected with EV71 at a m.o.i. of 40, and viral titers during the course of infection were titrated by plaque assays. Relative amounts of protein expression levels in each experimental set were also tested. The data were analyzed statistically by one-way ANOVA. Error bar: standard deviation; *: <i>p</i> <0.05 and **: <i>p</i> <0.01.</p

Confirmation of FBP1 cleavage at Gly-371 by EV71 2A^pro.

<p>(A) [³⁵S] methionine-labeled FBP1 and FBP1 fragments containing aa 1–371, 372–644, 1–443, 185–644, and 185–443 were treated (+) or untreated (-) with EV71 2A^pro (2A). (B) Schematic representation of FBP1, FBP1 fragments and the proposed primary cleavage site at Gly-371 (indicated by an arrow). The molecular masses of the corresponding cleavage products are also shown. (C) RD cells transfected with FLAG-HA dual-tagged FBP1 and mutant FBP1^G371K, the latter of which is resistant to 2A^pro cleavage, were infected with EV71. At 4, 6, 8 and 10 h.p.i., cell lysates were prepared and analyzed by immunoblotting with anti-FLAG, anti-HA, anti-EV71 3D^pol and anti-actin antibodies. Cleavage products containing the FLAG-tag or HA-tag are indicated as Cp. Cp-N and Cp-C: Cleavage products respectively containing the N-terminal or C-terminal region of FBP1.</p