Fig 1.
NDV infection induces the inhibition of host protein synthesis and eIF4F complex phosphorylation and assembly.
(A) The growth curve of NDV in HeLa cells. HeLa cells were infected with 5 MOI of Herts/33. Supernatants were harvested at indicated times and were subjected to TCID50 assay. TCID50 was calculated using Reed-Munch mathematical analysis. (B) HeLa cells infected with Herts/33 were labeled with 100 μCi of [35S] methionine/cysteine for 1 h and collected at the indicated time. Labeled proteins were analyzed by SDS-PAGE followed by fluorography and autoradiography. Asterisks (*) indicate newly synthesized proteins detected only in Herts/33 infected cells. Molecular weight standards appear in the leftmost lane and their sizes (kDa) are indicated in the margin. Coomassie brilliant blue staining of the autoradiograph gel were performed to confirm the equivalence of protein loading. (C) Quantitation of host protein synthesis in NDV-infected HeLa cells. The rates of protein synthesis were determined as fold changes of host protein synthesis in NDV-infected cells compared to that in mock-infected cells. (D) HeLa cells were mock-infected or infected with NDV, and harvested at indicated times. Total protein was isolated, and equivalent amounts were fractionated by SDS-PAGE, and analyzed by immunoblotting using antibodies recognizing phospho-eIF2α (p-eIF2α), total eIF2α, NP, and β-actin. (E) Phosphorylation of eIF4E and eIF4G is stimulated in NDV infected HeLa cells. HeLa cells were either mock-infected (Mock) or infected with NDV (5MOI). At the indicated times points, total protein was isolated, and equivalent amounts were fractionated by SDS-PAGE, and analyzed by immunoblotting using antibodies recognizing phospho-eIF4E (p-eIF4E), total eIF4E, phospho-eIF4G (p-eIF4G), total eIF4G, eIF4A, NP and β-action. (F) The phosphorylation status of 4E-BP1 was analyzed using antibodies against total 4E-BP1, phopho-4E-BP1 (Thr70), phopho-4E-BP1 (Ser65), phopho-4E-BP1 (Thr37/46) and non-phospho-4E-BP1 (Thr46). (G) eIF4F complex were precipitated from the lysates mentioned in (D) by m7GTP pull down assay. The precipitates were boiled in 2×loading buffer and the component proteins were analyzed by western blotting as shown. The band intensities of eIF4G and 4E-BP1 were determined and normalized to eIF4E. The intensity bind ratio for mock was given a value of 1 and indicated in the frames below the blots. (H) HeLa cells were transfected with siControl, sieIF4E, sieIF4G or a mixture of sieIF4E and sieIF4G (siMix) as indicated. After 48 h, cells were infected with 5 MOI of NDV and samples were recovered after 8 and 12 hpi respectively. Depletion of eIFs was examined by Western blotting against eIF4G and eIF4E. Accumulation of viral proteins was analyzed using specific antibodies against NP protein. The band intensities of eIF4G, eIF4E and NP were determined and normalized to β-actin. The intensity bind ratio for cells transfected with nontarget siRNA (siControl) was given a value of 1 and indicated in the frames below the blots. (I) Supernatants from transfected cells were collected at the indicated times and determined by TCID50 assay. Viral titers were expressed as percentage of control. Data are presented as means from three independent experiments. Significance was analyzed by one-way analysis of variance followed by Dunnett’s test (*, p<0.05; ns, not significant).
Fig 2.
NDV activates PI3K/Akt/mTOR pathway to phosphorylate downstream proteins that regulate translation in order to promote the eIF4F assembly and the production of NDV virus proteins.
(A) HeLa cells infected with 5MOI of NDV Herts/33 were harvested at the indicated times, both phosphorylation and total of Akt, mTOR, p70S6K and rpS6 were analyzed by western blotting. (B) PI3K specific inhibitor LY294002 suppressed Akt/mTOR/4E-BP1 phosphorylation induced by the virus infection. Cells preincubated with medium in the absence of LY294002 and then mock infected or infected with the virus were used as the negative and positive control, respectively. HeLa cells were pretreated with LY294002 and infected with NDV at an MOI of 5 in the presence of the inhibitor, and then processed for Western blotting at 8hpi with antibodies as shown. (C) eIF4G-eIF4E association and NDV protein synthesis are abrogated by LY294002 treatment. HeLa cells were pretreated with 20 μM LY294002. After 2 h cells were infected with NDV (5 MOI) in the continuous presence of the compounds. At 8 hpi cells were incubated with 7-Methyl GTP-Sepharose 4B beads. eIF4G, eIF4E, 4E-BP1, NP and β-actin were detected in eluted or full lysate by Western blotting. The band intensities of NP was determined and normalized to β-actin. The intensity bind ratio for infected cells in the absence of LY294002 was given a value of 1 and indicated in the frames below the blots. (D) The efficacy of cap Sepharose pull-down in the presence of LY294002 was determined using immunoblotting for eIF4E, eIF4G and 4E-BP1. The efficacies of eIF4G and 4E-BP1 pull-down are expressed and normalized as the percentage of the eIF4E. (E) The extracellular virus yields were determined by TCID50 assay at 8 hpi and expressed as percentage of control. Data are presented as means from three independent experiments. Significance is analyzed by two-tailed Student's t-test (*, p<0.05; ns, not significant).
Fig 3.
Phosphorylation of eIF4G requires mTOR activity and NDV mediated 4EBP1 phosphorylation is rapamycin resistant.
(A-B) NDV infection increases level of phosphorylated eIF4G through mTOR activation. HeLa cells were non-treated or pretreated for 2 h with rapamycin 100 nM (A) or LY294002 20 μM (B) then infected with 5MOI of NDV. Cells were then lysed with buffer sample at 8 hpi. Amounts of phospho-eIF4G and total eIF4G were analyzed by Western blotting. β-actin was detected as a load control. (C) Effect of NDV infection on total levels and phosphorylation status of mTOR effectors in the presence and absence of rapamycin. HeLa cells were pretreated 2h with 100nM rapamycin and infected or mock infected in the presence with rapamycin for 8h after which the cells were harvested, protein lysates prepared and phosphorylation of mentioned molecules were subsequently analyzed by immunobloting. (D) NDV induced 4E-BP1 phosphorylation is rapamycin insensitive. As in C, the phosphorylation status of 4E-BP1 was analyzed using antibodies against total 4E-BP1, phopho-4E-BP1 (Thr70), phopho-4E-BP1 (Ser65), phopho-4E-BP1 (Thr37/46) and non-phospho-4E-BP1 (Thr46). (E) Rapamycin treatment can’t suppress the eIF4F assembly and production of NDV virus proteins. HeLa cells were pretreated with 100 nM Rapamycin. After 2 h cells were infected with NDV (5 MOI) in the continuous presence of the compounds. At 8 hpi cells were incubated with 7-Methyl GTP-Sepharose 4B beads. eIF4G, eIF4E, 4E-BP1, NP and β-actin were detected in eluted or full lysate by Western blotting. The band intensities of NP was determined and normalized to β-actin. The intensity bind ratio for infected cells in the absence of Rapamycin was given a value of 1 and indicated in the frames below the blots. (F) The efficacy of cap Sepharose pull-down in the presence of Rapamycin was determined using immunoblotting for eIF4E, eIF4G and 4E-BP1. The efficacies of eIF4G and 4E-BP1 pull-down are expressed and normalized as the percentage of the eIF4E. (G) The extracellular virus yields were determined by TCID50 assay at 8 hpi and expressed as percentage of control. Data are presented as means from three independent experiments. Significance is analyzed by two-tailed Student's t-test (*, p<0.05; ns, not significant).
Fig 4.
Activation of p38 MAPK/ Mnk-1 pathway induces eIF4E phosphorylation, but is not essential for viral protein synthesis.
(A) Phosphorylation of p38, Erk1/2 and Mnk-1 is stimulated in NDV infected HeLa cells. HeLa cells were either mock-infected or infected with NDV (5MOI). At the indicated times (hpi), total protein was isolated, and equivalent amounts were fractionated by SDS-PAGE, and analyzed by immunoblotting using antibodies as shown. (B-D) eIF4E phosphorylation results from the activation of p38, but not Erk, in NDV-infected HeLa cells. Samples (as described in A) isolated from cultures treated with either DMSO, the p38 inhibitor SB203580 (B), the MEK1/2 inhibitor U0126 (C) or CGP 57380 (D) were analyzed by Western blotting as in (A). The band intensities of p-eIF4E was determined and normalized to eIF4E. The intensity bind ratio for uninfected cells in the absence of SB203580 (B) and U0126 (C) was given a value of 1 and indicated in the frames below the blots. (E) Phosphorylation of 4E-BP1 induced by NDV is not mediated by p38/Mnk-1 pathway. HeLa cells were pretreated 2 h with 10 μM SB203580 or CGP57380 and infected or mock-infected in the presence of inhibitors for 8 h before cells were harvested. The phosphorylation status of 4E-BP1 was analyzed using antibodies against total 4E-BP1, phopho-4E-BP1 (Thr70), phopho-4E-BP1 (Ser65), phopho-4E-BP1 (Thr37/46) and non-phospho-4E-BP1 (Thr46). (F) eIF4F complex loading is not affected by eIF4E dephosphorylation. eIF4F complexes were precipitated from protein lysates described in (A) by m7 GTP pull-down assay. eIF4G, eIF4E, 4E-BP1, NP and β-actin were detected in eluted or full lysate by Western blotting. The band intensities of NP was determined and normalized to β-actin. The intensity bind ratio for infected cells in the absence of CPG57380 was given a value of 1 and indicated in the frames below the blots. (G) The efficacy of cap Sepharose pull-down in the presence of CGP57380 was determined using immunoblotting for eIF4E, eIF4G and 4E-BP1. The efficacies of eIF4G and 4E-BP1 pull-down are expressed and normalized as the percentage of the eIF4E. (H) The extracellular virus yields were determined by TCID50 assay at 8 hpi and expressed as percentage of control. Data are presented as means from three independent experiments. Significance is analyzed by two-tailed Student's t-test (*, p<0.05; ns, not significant). (I) Mnk1 kinase was depleted in HeLa cells using siRNA. At 48 h post transfection, the cells were infected with NDV at 5 MOI in the presence of rapamycin or a DMSO solvent control and harvested at 8 hpi. Total protein was isolated, and equivalent amounts were fractionated by SDS-PAGE, and analyzed by immunoblotting using antibodies as shown. (J) Western blot analysis for eIF4F assembly of m7GTP precipitates HeLa cells that were infected with 5 MOI NDV in the presence of rapamycin or a DMSO solvent control after transfection with duplex siRNA oligonucleotides against MNK1 or control siRNA. NP and β-actin were detected in full lysate by Western blotting. The band intensities of NP was determined and normalized to β-actin. The intensity bind ratio for mock cells was given a value of 1 and indicated in the frames below the blots. (K) The efficacy of cap Sepharose pull-down was determined using immunoblotting for eIF4E, eIF4G and 4E-BP1. The efficacies of eIF4G and 4E-BP1 pull-down are expressed and normalized as the percentage of the eIF4E. (L) The extracellular virus yields were determined by TCID50 assay at 8 hpi and expressed as percentage of control. Data are presented as means from three independent experiments. Significance is analyzed by one-way analysis of variance followed by Dunnett’s test (*, p<0.05; ns, not significant).
Fig 5.
NDV NP protein physically interacts with eIF4E.
(A) m7GTP pull-down of NP with eIF4F complex. HeLa cells infected with 5MOI of NDV Herts/33 were harvested at 8 hpi, lysates prepared and eIF4F complex were precipitated by m7GTP pull down assay. The precipitates were boiled in 2×loading buffer and the component proteins were analyzed by western blotting for the detection of P, NP, eIF4E, eIF4G 4E-BP1 and β-actin. (B) Association of NP with eIF4F in vitro. HeLa cells were transfected with the indicated plasmids or empty vectors, and the whole cell lysates obtained at 48 hpi. Proteins precipitated with m7GTP sepharose beads were detected by immunobloting with specific antibodies against FLAG, eIF4E, eIF4G 4E-BP1 and β-actin. (C) 293T cells were transfected with 3×FLAG tagged eIF4E or eIF4G and HA-tagged NP. These cells were harvested after transfection for 48 h. The interaction between NP and eIF4E was confrmed by co-IP with an anti-FLAG antibody and immunoblotting with an anti-HA antibody. (D) Reciprocal co-precipitation of NP with endogenous eIF4E. Cell lysates expressing FLAG-NP were immunoprecipitated with an FLAG or eIF4E antibodies and immunobloted with specific antibodies as mentioned. (E) Co-IP of NP protein with endogenous eIF4E during NDV infection. NDV-infected (+) or mock-infected (-) HeLa cells were used for IP with anti-NP protein or eIF4E antibody and immunoblotted with the indicated antibodies. (F) GST pulldown assay. Glutathione beads conjugated to GST or the GST-NP fusion protein were incubated with eIF4E overexpressing cell lysate. After washing, proteins were eluted from the beads and SDS-PAGE was performed. The presence of eIF4E was detected by immunoblotting with anti-Flag antibody. GST and GST-NP protein expression was confirmed by immunoblotting with anti-GST antibody. (G) eIF4E was redistributed and colocalized with NDV NP protein.HeLa cells were seeded on glass coverslips and mock-infected or infected with NDV Herts/33 at an MOI of 5. At 8 hpi cells were fixed and stained with anti-eIF4E and NP antibodies and then visualized by confocal microscopy. (H) Schematic representation of the deletion mutants of NP protein. Square frames represent the protein product of each truncated NP gene and the amino acid positions are indicated upon the frames. Dotted lines indicate deleted regions. (I, J) The N-terminal 391 residues of NP are sufficient to allow a heterologous protein to associate with eIF4E. Multiple C-terminal and N-terminal truncations of Flag-tagged NP were expressed in HeLa cells respectively. eIF4E were isolated by adsorption to 7-methyl GTP Sepharose beads, fractionated by SDS-PAGE, and analyzed by immunoblotting with the specific antibody.
Fig 6.
NDV NP protein fractionates with polysomes and inhibits protein synthesis in vitro and in vivo.
(A) Upper panel: total cytoplasmic ribosomes (from lysates of NDV-infected and mock-infected HeLa cells) were fractionated by sucrose density gradient centrifugation. The ribosome profiles were obtained by measuring the absorbance at 254 nm of individual fractions. Cycloheximide was present in each sample. Lower panels: fractions were immunoblotted using the antibodies indicated on the right. (B) Ribosome profiles of NDV-infected and mock-infected HeLa cell extracts treated with 20mM EDTA to dissociate polysomes. Fractions were immunoblotted using the antibodies indicated on the right. (C) Distribution of β-actin mRNA with ribosomal complexes in NDV-infected HeLa cells. Transcript number, as determined by quantitative RT-PCR, is expressed as the percentage of total β-actin transcripts recovered and plotted against fraction number. The results are given as the mean ± SD from a representative quantitative RT-PCR experiment performed in duplicate. (D) Distribution of NDV NP mRNA with ribosomal complexes in NDV-infected HeLa cells treated with 20mM EDTA. Lysates were resolved and fractionated, and mRNA distribution was determined as in C. (E, F) Increasing amounts of recombinant GST (E) and GST-NP (F) were incubated with rabbit reticulocyte lysates. A capped reporter, RNA encoding the Renilla luciferase was added and translated. Translation products were detected by measuring the activity of Renilla luciferase and Western blot analysis. The translation efficiency of Renilla luciferase is expressed as a percentage of the control set to 100%. Results show the means±S.E. from three separate experiments. Significance was analyzed by one-way analysis of variance followed by Dunnett’s test (*, p<0.05; ns, not significant). The intensities of Renilla luciferase bands were quantified by densitometric analysis and indicated below the blots. (G) A reporter mRNA was preincubated with increasing amount of GST-NP and then added to rabbit reticulocyte lysates. Translated products were quantified in a similar manner as panel E and F. (H) NDV-NP protein was expressed in HeLa cells by lentiviral packaging system and the expression of NP was detected by Western blotting. (I, J) A reporter plasmid encoding the Renilla luciferase gene was transfected with increasing concentrations in HeLa-GFP and HeLa-NP cells respectively. Cells were harvested, an aliquot was lysed, and Renilla luciferase activity was measured by using a luminometer (I). The relative luciferase activity was indicated in the frames below the graphics. Another aliquot was harvested, and the total RNA was analyzed for Renilla luciferase transcripts using real-time RT-PCR. The Renilla luciferase mRNA levels in HeLa-NP cells are expressed relative to those in HeLa-GFP cells (J). The results shown represent means of three experiments. Significance is analyzed by two-tailed Student's t-test (*, p<0.05; ns, not significant).
Fig 7.
Proposed model for the cap-dependent translational control during NDV infection.
NDV infection activates the host cap-dependent translation machinery to benefit viral translation and replication. NDV enhances eIF4E and eIF4G phosphorylation to promote eIF4F assembly and accelerate translation initiation by concomitant upregulation of PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways. Activated mTOR enhances translation initiation machinery through phosphorylation of the translational repressor 4E-BP1 and S6K1/rpS6. rpS6 phosphorylation may contribute to the sustained translation of host 5’TOP mRNAs which encode ribosomal protein, thereby facilitating the production of progeny viruses by supporting viral mRNA translation. In addition to phosphorylate eIF4E, p38 MAPK/Mnk1 signaling pathway also maintians 4EBP1 hyperphosphorylation and vrial protein synthesis to be resistant to rapamycin treatment. Finally, NDV NP protein can bind to eIF4E and facilitate the selective translation of viral mRNAs based on the involvement in cap-dependent translation during NDV infection by associating with polysomes and the ability to suppress host mRNA translation in vitro and in vivo.