Bovine Adenovirus-3 pVIII Suppresses Cap-Dependent mRNA Translation Possibly by Interfering with the Recruitment of DDX3 and Translation Initiation Factors to the mRNA Cap

Abstract

Earlier, targeting of DDX3 by few viral proteins has defined its role in mRNA transport and induction of interferon production. This study was conducted to investigate the function of bovine adenovirus (BAdV)-3 pVIII during virus infection. Here, we provided evidence regarding involvement of DDX3 in cap dependent cellular mRNA translation and demonstrated that targeting of DDX3 by adenovirus protein VIII interfered with cap-dependent mRNA translation function of DDX3 in virus infected cells. Adenovirus late protein pVIII interacted with DDX3 in transfected and BAdV-3 infected cells. pVIII inhibited capped mRNA translation in vitro and in vivo by limiting the amount of DDX3 and eIF3. Diminished amount of DDX3 and eIFs including eIF3, eIF4E, eIF4G, and PABP were present in cap binding complex in BAdV-3 infected or pVIII transfected cells with no trace of pVIII in cap binding complex. The total amount of eIFs appeared similar in uninfected or infected cells as BAdV-3 did not appear to degrade eIFs. The co-immunoprecipitation experiments indicated the absence of direct interaction between pVIII and eIF3, eIF4E, or PABP. These data indicate that interaction of pVIII with DDX3 interferes with the binding of eIF3, eIF4E and PABP to the 5' Cap. We conclude that DDX3 promotes cap-dependent cellular mRNA translation and BAdV-3 pVIII inhibits translation of capped cellular mRNA possibly by interfering with the recruitment of eIFs to the capped cellular mRNA.

Keywords: BAdV-3; DDX3; cap-dependent mRNA translation; pVIII.

FIGURE 1

Interaction of DDX3 with BAdV-3 pVIII. (A) Glutathione S-transferase (GST) pull down assay. Purified GST or GST.pVIII fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated with in vitro translated [³⁵S] methionine labeled HA tagged DDX3 were separated by 10% SDS-PAGE and detected by autoradiography. (B,C) Co-immunoprecipitation in transfected cells. Proteins from the lysates of cells co-transfected with either pHA.DX3 and pEY.pVIII or pHA.DX3 and pEYFPN1 were immunoprecipitated with anti-pVIII serum (B) or anti-HA MAb (C), separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The separated proteins were probed in Western blot using anti-HA MAb (B) or anti-pVIII serum (C). (D) Co-immunoprecipitation in BAdV-3 infected cells. Proteins from the lysates of mock or BAdV-3 infected Madin-Darby Bovine Kidney (MDBK) cells were immunoprecipitated with anti-pVIII serum, separated by 10% SDS-PAGE, transferred to nitrocellulose membrane and probed in Western blot using anti-DDX3 MAb. Immunoprecipitation (IP). WB (Western blot). Ctl (Control). (E–G) Confocal microscopy. MDBK cells mock infected (panels a and f) or infected with BAdV-3 (panels d and g1–g4) VERO cells untransfected (panel b) or transfected with indicated plasmid (panels c, e, and h1–h4) DNA, were fixed 36 h post-infection/transfection. The subcellular localization of DDX3 (panels a–c, g2, and h2) protein was visualized by indirect immunofluorescence (panels a–c, g2, h2) using anti-DDX3 MAb and fluorescein conjugated goat anti-mouse IgG-FITC (panels a and g2), anti-DDX3 MAb and Cy3 conjugated goat anti-mouse (pane b) secondary antibody, anti-HA MAb and Cy3 conjugated goat anti-mouse secondary antibody (panel c and h2). The subcellular localization of pVIII (panels d, e, f, g1, and h1) was visualized by direct fluorescence (panels e and h1) or indirect immunofluorescence using anti-pVIII serum and Cy3 conjugated goat anti-rabbit secondary antibody (panels d, f, and g1). Nuclei were stained with DAPI in each panel. A merge of the images is shown. Enlargement of panel g4 and h4 is shown, arrows in white shows few of the colocalization of pVIII and DDX3.

FIGURE 2

Interaction of DDX3 with PAdV-3 and HAdV-5 pVIII. (A) Coomassie blue staining of purified protein. Purified GST.DDX3 protein was separated by 10% SDS-PAGE and stained with 0.25 Coomassie blue stain. (B) GST-pull down assay. Purified GSTor GST.DDX3 fusion protein immobilized on Glutathione-Sepharose 4B beads, incubated individually with in vitro translated [³⁵S] methionine labeled PAdV-3 pVIII or HAdV-5 pVIII, separated by 10% SDS-PAGE and detected by autoradiography. (C) Co-immunoprecipitation. Radio labeled in vitro transcribed and translated HAdV5 pVIII or PAdV-3 pVIII was incubated with in vitro transcribed and translated unlabeled DDX3 protein. Proteins were immunoprecipitated with either anti-DDX3 serum or rabbit pre immune sera, separated by 10% SDS-PAGE and auto radio-graphed. Immunoprecipitation (IP).

FIGURE 3

Protein synthesis in BAdV-3 infected cells. (A) Monolayers of MDBK cells were mock infected or infected with BAdV-3 at a MOI of 5. (B,C) Monolayers of VERO cells were transfected with indicated amounts of plasmid DNAs. At indicated times post infection (A) or transfection (B,C), the cells were pulse labeled with [³⁵S] methionine for 10 min. The radiolabelled proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. Proteins from the lysates of radiolabeled cells (A,B or C) were subjected to SDS-PAGE and Western blot using anti-pVIII serum, or anti-β-actin MAb.

FIGURE 4

Effect of pVIII on capped mRNA translation. (A). In vitro. The TNT^® T7 luciferase DNA (Promega) (i) was transcribed in vitro in the absence (uncapped) or presence (capped) of 40 mM Ribo m7GpppG cap analog (Promega) using RiboMAX RNA production system-T7 (Promega). The in vitro synthesized capped and uncapped luciferase mRNAs (ii) were translated in the supernatant collected after centrifugation of mixture of Flexi Rabbit Reticulo Lysate (Promega) incubated with Glutathione sepharose beads preloaded with GST.VIII or GST protein alone. The level of luciferase activity was measured using a luciferase kit (Promega) on a Luminometer (Turner Designs, Inc.). The results are shown as relative luciferase activity (iii). Error bars indicate SE of means for separate experiments. The relative luciferase intensity is determined based on GST compared to GST.pVIII. (B) In vivo. 293T cells were transfected with plasmid DNAs (2 μg of pcDNA3-RLuc-POLIRES-FLuc (i) and either 4 μg of pEY.pVIII or 4 μg of pEYFPN1). At 36 h post transfection, Firefly luciferase (FLuc) and Renilla reniformis luciferase (RLuc) activities were measured in a luminometer by using a dual luciferase assay kit (Promega) as per the company’s procedure. Expression of EYFP was used to normalize the transfection efficiency. The results are shown as relative luciferase activity (iii). The level of cytoplasmic RLuc-POLIRES-FLuc mRNA both in EY.pVIII and EYFP expressing plasmid transfected cells was quantified by RT-PCR (ii). Error bars indicate SE of means for three separate experiments. ^∗statistically significant.

FIGURE 5

The effect of pVIII on levels of eIFs. The cytoplasmic fraction of MDBK cells (50 μl) or Flexi Rabbit Reticulo-Lysate (10 μl) were incubated with either 10 μl of GST beads loaded with 750 ng of purified GST.VIII protein or 10 μl of GST beads loaded with 750 ng of purified GST protein for 2 h at +4°C and centrifuged for 10 min. The supernatant (S) and the pellet (P) from both reticulo lysate (A) or cytoplasmic fraction (B) were separated by 10% SDS-PAGE gel and analyzed by Western blot using indicated protein specific antibodies and Alexa Flour 680 goat anti-rabbit IgG antibody or IRDye 800 conjugated goat anti-mouse IgG as secondary antibody.

FIGURE 6

m7GTP-sepharose binding assay. (A) The supernatant of the lysates of the cells collected at 36 h post BAdV-3 infection of MDBK cells (mock or BAdV-3) or transfection of 293T cells with plasmid DNAs (pEY.pVIII or pEYFPN1) were incubated with m7GTP sepharose cap affinity beads. After washing, the bound proteins were analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. The intensity of the bands of the Western blot in all cases was analyzed by Odyssey Software v2.1. The relative amount of proteins in BAdV-3 infected or pEY.VIII transfected cell lysates that are retained in the 7-methyl GTP resins as compared to mock infected or pEYFPN1 transfected cells, respectively (i.e., considering the amount of protein in mock infected or pEYFPN1 transfected cell lysates that are retained in the m7GTP resins as 100%) is plotted. Error bars indicate SE of means for three separate experiments. Proteins from the lysates of BAdV-3 infected or transfected cells were separated by 10% SDS-PAGE and probed in Western blot using anti-pVIII serum. (B) Proteins from the lysates of mock infected or BAdV-3 infected MDBK cells collected at 36 h post infection were separated by 10% SDS-PAGE and analyzed by Western blot using protein specific antibody and anti-rabbit IRDye 800 conjugated goat anti-mouse IgG (Li-COR biosciences) or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. β-actin was used as a loading control.

FIGURE 7

Interaction of pVIII with eIFs. (A) The cytoplasmic fraction (50 μl) of DDX3 positive or negative VIDO R2 cells were incubated with 10 μl of beads loaded with 750 ng of purified GST-VIII protein for 2 h at 4°C and centrifuged for 10 min. The supernatant (S) and the pellet (P) from both cytoplasmic fraction were separated by 10% SDS-PAGE gel and analyzed by Western blot using indicated protein specific antibodies and IRDye 800 conjugated goat anti-mouse IgG or Alexa Flour 680 goat anti-rabbit IgG as secondary antibody. Total amount of indicated protein in cytoplasmic fraction of indicated cells was estimated by Western blot analysis before adding GST.pVIII fusion protein. (B) DDX3 knockdown 293T cells were transfected with 2 μg of pHA.DX3 or pHA.pVIII or pHA.DX3 and pc.pVIII. The cells were lysed at 48 h post transfection and precipitated by anti-HA affinity matrix and the precipitates were analyzed by Western blot as indicated above.

FIGURE 8

Protein synthesis in BAdV-3 infected DDX3kd VIDOR2 cells. (A) Western blot of lysates of DDX3 +Ve or DDX3kd VIDOR2 cells with anti-DDX3 antibody. (B,C) [³⁵S] methionine pulse labeling and quantification. Mock infected or BAdV-3 infected cells were pulse labeled with [³⁵S] methionine for 10 min at 36 h or 48 h post infection. The radiolabelled proteins were separated by 10% SDS-PAGE and analyzed by autoradiography. (C) The intensity of each lane was quantified using GelQuant.NET software and plotted relative to the intensity of the mock infected cell lanes.

FIGURE 9

qRT-PCR. MDBK cells were infected with BAdV-3 at an MOI of 5. At indicated times post infection cytoplasmic RNA was purified and cDNA synthesized and qRT-PCR performed using specific primers targeting the indicated bovine housekeeping genes as described in the materials and methods. Western blot was performed using anti-BAdV-3 DBP (DNA binding protein) to confirm productive infection. Error bars indicate S.E of means for three separate experiments.