Journal: PLOS Biology

Article Title: The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

doi: 10.1371/journal.pbio.3001693

Figure Lengend Snippet: (A) Start codons utilized by eIF2, eIF2A, and eIF2D initiation factors . Amino acids for each codon is in parentheses. Initiation factor-specific start codons are indicated in red. (B) Comparison of infectious virus produced by the indicated donor RNAs with modifications that alter the conserved AUG to initiation factor specific start codons: ①: ΔIRES; ②: ΔAUG; ③: AUG to UUG; ④: AUG to UUU; ⑤: AUG to CUG; ⑥: AUG to CUA; ⑦: AUG to CUC. Codons UUG, UUU, and CUG can be utilized by initiation factors, but CUA and CUC cannot. In all cases, the acceptor RNA contained two STOP codons after 3B-coding sequence (3B STOP) and the mutation that inactivates the RdRp (GDD to GAA). The indicated set of donor and acceptor RNAs in a 1:5 molar ratio (total 0.3 μg) was cotransfected into HAP1 WT cells. Indicated are the relative viral titers with the average viral titer from recombination using ΔIRES donor (①) and acceptor set as 100% (647 pfu/mL, mean ± SEM; n = 3). Statistical analyses were performed using unpaired, two-tailed t test (*significance level p = < 0.05, ** p = < 0.01, n.s. indicates not significant). The ΔAUG, CUA, and CUC reduced viral recombinants significantly, while UUG, UUU, and CUG did not, consistent with eIF2A and eIF2D contributing to IRES-independent translation. Numerical data provided as Supporting information . (C, D) Viral recombinants are reduced in HAP1 cells deficient for eIF2A and eIF2D expression. The indicated set of donor and acceptor RNAs was cotransfected into HAP1 WT or eIF2A-KO or eIF2D-KO cells. Donor RNA: ΔIRES (panel C ); RLuc-WT (panel D ). Relative viral titers with the average viral titer from HAP1 WT set as 100% were shown (panel C , 7,433 pfu/mL; panel D , 243 pfu/mL; mean ± SEM). Statistical analyses were performed using unpaired, two-tailed t test (**** indicates p < 0.0001). Numerical data provided as Supporting information . IRES, internal ribosome entry site; KO, knockout; RdRp, RNA-dependent RNA polymerase; WT, wild-type.

Article Snippet: Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech.

Techniques: Produced, Sequencing, Mutagenesis, Two Tailed Test, Expressing, Knock-Out

Journal: PLOS Biology

Article Title: The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

doi: 10.1371/journal.pbio.3001693

Figure Lengend Snippet: (A) Nucleotide substitutions at each position across the EV-A71 genome have been defined by deep RNA sequencing. The average substitution frequency is 38. Some regions exhibit a lower average; these regions of the genome may encode cis -acting RNA elements. Numerical data provided as Supporting information . The entire data set is available at the GEO repository under accession number GSE183959. (B) The region from 3,602–3,679 of the EV-A71 genome exhibits a below-average frequency of nucleotide substitutions. This region corresponds to a sequence 78-nt upstream of the AUG used for eIF2A/2D-dependent translation. Numerical data provided as Supporting information . (C) Alignment of the corresponding nucleotide sequence and amino acid sequence reveals moderate to high sequence identity among enterovirus (PV, EV-A71, CVB3, and EV-D68). Black asterisks indicate conservation across all enteroviruses; red asterisks indicate conservation in three of the four enteroviruses. (D) RNA secondary structure is predicted in this region for all enteroviruses. The RNAfold algorithm was used. The details of the fold varied across the enteroviruses more substantially than sequence might predict. (E) Comparison of infectious PV produced using donor RNAs harboring a deletion of the 78-nt sequence (Δ78 nt) or containing the corresponding sequences from EV-A71 or EV-D68. The acceptor RNA used does not support translation of 3CD or an active polymerase. Virus produced (pfu/mL ± SEM; n = 3) from each cotransfection is shown. Statistical analyses were performed using unpaired, two-tailed t test (* indicates p < 0.05, ** indicates p < 0.01; n.s. indicates nonsignificant). The sequence contributes to production of virus. Numerical data provided as Supporting information . (F) Contribution of the 78-nt sequence to IRES-independent translation indirectly by monitoring PI4P levels and localization as described in the legend to . Donor RNAs of panel E were used, in addition to the ΔAUG donor RNA used in that has a strong defect to virus production. Quantitation of PI4P staining in approximately 60 cells selected randomly from three separate fields expressed as fluorescence intensity is shown. A significant reduction of PI4P/translation of the genome ( p < 0.0001) was observed for Δ78 and ΔAUG only. Scale bars are equivalent to 10 μm. Numerical data provided as Supporting information . GEO, Gene Expression Omnibus; IRES, internal ribosome entry site; PI4P, phosphatidylinositol-4-phosphate; PV, poliovirus.

Article Snippet: Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech.

Techniques: RNA Sequencing Assay, Sequencing, Produced, Cotransfection, Two Tailed Test, Quantitation Assay, Staining, Fluorescence, Expressing

Journal: PLOS Biology

Article Title: The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

doi: 10.1371/journal.pbio.3001693

Figure Lengend Snippet: (A) NanoLuc activity in HAP1 WT cells transfected with a full-length PV genome with the nanoLuc-coding sequence embedded between 2C- and 3A-coding regions (2C/3A-Nluc). As a control, the PV 2C/3A-Nluc RNA was transfected in the presence of GuHCl. Numerical data provided as Supporting information . (B) Comparison of nanoLuc activity at six hours posttransfection using PV 2C/3A-Nluc RNA in HAP1 WT, eIF2A-KO, and eIF2D-KO cells. Data from one of two biological replicates with similar results, each with two technical replicates. Numerical data provided as Supporting information . (C) Comparison of nanoLuc activity at six hours postinfection using an MOI of 0.1 or 1 in HAP1 WT, eIF2A-KO, and eIF2D-KO cells. Data from one of two biological replicates with similar results, each with two technical replicates. Numerical data provided as Supporting information . (D) Comparison of nanoLuc activity at six hours posttransfection using PV 2C/3A-Nluc RNA (Transfection), and at six hours postinfection using an MOI of 0.1 or 1 (Infection) in HAP1 WT, and eIF2A-KO/eIF2D-KO cells. Data from one of two biological replicates with similar results, each with two or three technical replicates. Statistical analyses were performed using unpaired, two-tailed t test (** indicates p < 0.01, n.s. indicates not significant). Numerical data provided as Supporting information . (E-G) Western blot analysis of eIF2A (panel E ), eIF2D (panel F ), and eIF2α (panel G ) in HAP1 WT, eIF2A-KO, eIF2D-KO, and eIF2A-KO/eIF2D-KO cells. Cells were processed for western blot analysis and probed using anti-eIF2A, eIF2D, and eIF2α antibodies. GAPDH and tubulin were used as a loading control for western blot. Blots provided in Supporting information . GuHCl, guanidine hydrochloride; KO, knockout; PV, poliovirus; RLU, relative light unit; WT, wild-type.

Article Snippet: Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech.

Techniques: Activity Assay, Transfection, Sequencing, Infection, Two Tailed Test, Western Blot, Knock-Out

Journal: PLOS Biology

Article Title: The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

doi: 10.1371/journal.pbio.3001693

Figure Lengend Snippet: Single-cell analysis [ , ] using an MOI of 5 of PV-eGFP PV in HAP1 WT, eIF2A-KO, and eIF2D-KO cells. Comparison of the distributions of each parameter: start point (panel A ); maximum (panel B ); slope (panel C ); and infection time (panel D ); is shown (E) Percentage of PV-infected cells using HAP1 WT, eIF2A-KO, and eIF2D-KO cells (mean ± SD, n = 3) (Left). Adjusted P values of the t tests (Right). (F, G) Quantitative analysis from the data presented in panels A - D . Shown are the mean and standard deviation for each of the indicated parameters (panel F ). Adjusted P values of the t tests (panel G ). Numerical data provided as Supporting information . hpi, hours postinfection; KO, knockout; NFI, normalized fluorescence intensity; PV, poliovirus; WT, wild-type.

Article Snippet: Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech.

Techniques: Single-cell Analysis, Infection, Standard Deviation, Knock-Out, Fluorescence

Journal: PLOS Biology

Article Title: The enterovirus genome can be translated in an IRES-independent manner that requires the initiation factors eIF2A/eIF2D

doi: 10.1371/journal.pbio.3001693

Figure Lengend Snippet: (A) Under normal conditions, eIF4G and eIF4A bind to the primary IRES and recruit the 43S preinitiation complex composed of the 40S ribosomal subunit (yellow), eIF2/GTP/Met-tRNAi Met (green), and eIF3, among other factors. Initiation may also be facilitated by interactions of the poly(rA)-binding protein, PABP, bound to the 3′-poly(rA) tail. This interaction may use eIF4G or viral/cellular factors interacting with the cloverleaf. Translation begins at the AUG start site in an eIF2-directed manner. (B) Multiple distinct eIF2-independent mechanisms exist. Both eIF5B and MCT-1•DENR (brown) can substitute for eIF2 to promote recruitment of initiator tRNA and translation initiation at the AUG start site. These factors may also initiate at non-AUG codons. (C) A canonical IRES-independent, eIF2A/eIF2D-dependent mechanism. Activation of intrinsic antiviral defense mechanisms, for example, as a result of the presence of the 5′-OH as a component of structured RNA in our biosynthetic enteroviral genomes, will lead to inactivation of eIF2 and perhaps even sequestration of eIF3. The noncanonical translation initiation factors eIF2A and eIF2D (black) direct translation initiation from a region of RNA (red) within 2A-coding sequence in a manner that is not dependent on the presence of an AUG codon but may require cis -acting RNA element upstream of the site of translation initiation as suggested by the studies reported herein. Other than the 40S ribosomal subunit, factors leading to formation of the translation–initiation complex are not known.

Article Snippet: Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech.

Techniques: Binding Assay, Activation Assay, Sequencing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Betulinic Acid Suppresses Breast Cancer Metastasis by Targeting GRP78-Mediated Glycolysis and ER Stress Apoptotic Pathway

doi: 10.1155/2019/8781690

Figure Lengend Snippet: GRP78 overexpression suppresses aerobic glycolysis by activating PERK signaling to inhibit β -catenin. (a) Western blotting analysis verified that BA significantly enhanced GRP78 expression. (b) Overexpressed GRP78 led to the downregulation of c-Myc and subsequently decreased LDHA and p-PDK1/PDK1 but increased LDHB expression. (c) On the contrary, GRP78 knockdown reversed the inhibition of c-Myc, LDHA, and p-PDK1/PDK1 and the enhancement of LDHB induced by BA. (d) ISRIB (100 nM), the specific PERK inhibitor, inhibited eIF2 α phosphorylation and reversed β -catenin inhibition induced by BA. (e) Like BA, salubrinal (75 μ M) inhibited eIF2 α dephosphorylation and therefore downregulated β -catenin expression. (f) Coimmunoprecipitation assay revealed the binding of GRP78 and PERK, which was disrupted by BA in a dose-dependent manner (the results were obtained from triplicate experiments and were represented as mean values ± SD; ∗ P < 0.05 and ∗∗ P < 0.01).

Article Snippet: The primary antibodies included E-cadherin antibody (20874-1-AP, Proteintech, Rosemont, IL, USA), N-cadherin antibody (22018-1-AP, Proteintech, Rosemont, IL, USA), vimentin antibody (10366-1-AP, Proteintech, Rosemont, IL, USA), MMP-2 antibody (A6247, ABclonal Technology Cambridge, Boston, USA), MMP-9 antibody (sc-13520, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β -actin antibody (4970, Cell Signaling Technology, Danvers, MA, USA), β -catenin antibody (51067-2-AP, Proteintech, Rosemont, IL, USA), c-Myc antibody (A1309, ABclonal Technology Cambridge, Boston, USA), LDHA antibody (3582, Cell Signaling Technology, Danvers, MA, USA), LDHB antibody (sc-100775, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PDK-1 antibody (sc-293160, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-PDK-1 antibody (3061, Cell Signaling Technology, Danvers, MA, USA), GRP78 antibody (11587-1-AP, Proteintech, Rosemont, IL, USA), caspase-12 antibody (55238-1-AP, Proteintech, Rosemont, IL, USA), CHOP antibody (15204-1-AP, Proteintech, Rosemont, IL, USA), PERK antibody (5683, Cell Signaling Technology, Danvers, MA, USA), p-PERK antibody (DF7576, Affinity Biosciences, Cincinnati, OH, USA), eIF2 α (11233-1-AP, Proteintech, Rosemont, IL, USA), and p-eIF2 α (AP0635, ABclonal Technology Cambridge, Boston, USA).

Techniques: Over Expression, Western Blot, Expressing, Inhibition, De-Phosphorylation Assay, Co-Immunoprecipitation Assay, Binding Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Betulinic Acid Suppresses Breast Cancer Metastasis by Targeting GRP78-Mediated Glycolysis and ER Stress Apoptotic Pathway

doi: 10.1155/2019/8781690

Figure Lengend Snippet: Diagram illustrating how BA inhibited glycolysis by targeting GRP78. BA interrupted the binding of GRP78 and PERK, which initiated ER stress, and subsequently activated eIF2 α phosphorylation, resulting in β -catenin inhibition and c-Myc-mediated aerobic glycolysis. Meanwhile, the ER stress apoptotic pathway was triggered.

Article Snippet: The primary antibodies included E-cadherin antibody (20874-1-AP, Proteintech, Rosemont, IL, USA), N-cadherin antibody (22018-1-AP, Proteintech, Rosemont, IL, USA), vimentin antibody (10366-1-AP, Proteintech, Rosemont, IL, USA), MMP-2 antibody (A6247, ABclonal Technology Cambridge, Boston, USA), MMP-9 antibody (sc-13520, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β -actin antibody (4970, Cell Signaling Technology, Danvers, MA, USA), β -catenin antibody (51067-2-AP, Proteintech, Rosemont, IL, USA), c-Myc antibody (A1309, ABclonal Technology Cambridge, Boston, USA), LDHA antibody (3582, Cell Signaling Technology, Danvers, MA, USA), LDHB antibody (sc-100775, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PDK-1 antibody (sc-293160, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-PDK-1 antibody (3061, Cell Signaling Technology, Danvers, MA, USA), GRP78 antibody (11587-1-AP, Proteintech, Rosemont, IL, USA), caspase-12 antibody (55238-1-AP, Proteintech, Rosemont, IL, USA), CHOP antibody (15204-1-AP, Proteintech, Rosemont, IL, USA), PERK antibody (5683, Cell Signaling Technology, Danvers, MA, USA), p-PERK antibody (DF7576, Affinity Biosciences, Cincinnati, OH, USA), eIF2 α (11233-1-AP, Proteintech, Rosemont, IL, USA), and p-eIF2 α (AP0635, ABclonal Technology Cambridge, Boston, USA).

Techniques: Binding Assay, Inhibition

Journal: Pharmaceutical Biology

Article Title: Cinobufagin induces acute promyelocytic leukaemia cell apoptosis and PML-RARA degradation in a caspase-dependent manner by inhibiting the β-catenin signalling pathway

doi: 10.1080/13880209.2022.2118792

Figure Lengend Snippet: CBG stimulated ER stress. (A, B) NB4 and NB4-R1 cells were treated with different concentrations of CBG for 24 h, western blotting detects the PERK-eIF2α-ATF4 expression. (C, D) RT-PCR was used to measure the CHOP expression. Results are expressed as mean ± SD (n = 3). Compared with solvent group, * P < 0.05, ** P < 0.01, *** P < 0.001. (E, F) NB4 and NB4-R1 cells were incubated with 60 nM CBG, and immunofluorescence was performed to observe CHOP protein. Results are expressed as mean ± SD (n = 3).

Article Snippet: CBG (purity ≥98%, B20542) was purchased from Yuanye Biotechnology Co., LTD. (Shanghai, China), Z-VAD-FMK (A1902) and cycloheximide (CHX, A8244) were purchased from ApexBio (Houston, TX, USA), Suc-Leu-Leu-Val-Tyr-AMC (ab142120) and PML-RARA antibody (AB43152) were purchased from Abcam (Cambridge, MA, USA), lithium chloride (LiCl, L9650) was purchased from Sigma-Aldrich (St. Louis, MO, USA), cleaved caspase-3 (9661 T) and β-catenin (8480S) antibodies were purchased from Cell Signalling Technology (Danvers, MA, USA), microtubule-associated protein light chain 3 (LC3, 4600-1-AP), p62 (18420-1-AP), Bax (50599-2-Ig), Bcl-2 (12789-1-AP), Pro-caspase 3 (19677-1-AP), cyclin D1 (60186-1-Ig), c-myc (10828-1-AP), C/EBP homologous protein (CHOP, 60304-1-Ig), eukaryotic initiation factor 2α (eIF2α, 66482-1-Ig), protein kinase R-like endoplasmic reticulum kinase (PERK, 24390-1-AP), and β-actin (20536-1-AP) antibodies were purchased from Proteintech (Chicago, IL, USA), and p-eIF2α (Ser51) (AF3087), p-PERK (Thr982) (DF7576), and activating transcription factor 4 (ATF4, DF6008) antibodies were purchased from Affinity Biosciences (Cincinnati, OH, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Immunofluorescence