Newcastle Disease virus infection activates PI3K/Akt/mTOR and p38 MAPK/Mnk1 pathways to benefit viral mRNA translation via interaction of the viral NP protein and host eIF4E
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.