EBNA3C interacts with Gadd34 and counteracts the unfolded protein response

Abstract

EBNA3C is an EBV-encoded nuclear protein, essential for proliferation of EBV infected B-lymphocytes. Using EBNA3C amino acids 365-545 in a yeast two hybrid screen, we found an interaction with the Growth Arrest and DNA-damage protein, Gadd34. When both proteins are overexpressed, Gadd34 can interact with EBNA3C in both nuclear and cytoplasmic compartments. Amino acids 483-610 of Gadd34, including the two PP1a interaction, and the HSV-1 ICPgamma34.5 homology domains, are required for the interaction. Furthermore, interaction is lost with a mutant of EBNA3C (509 DVIEVID 515-->AVIAVIA), that abolishes EBNA3C coactivation ability as well as SUMO interaction1. In B-cells, Gadd34, and EBNA3C are present in a complex with PP1a using microcystin sepharose affinity purification, Using a lymphoblastoid cell line in which EBNA3C protein levels are conditional on hydroxytamoxifen, surprisingly, we found that (i) EBNA3C maintains phosphorylation of eIF2alpha at serine 51, and (ii) protects against ER stress induced activation of the unfolded protein response as measured by XBP1 (u) versus XBP1(s) protein expression and N-terminal ATF6 cleavage. In reporter assays, overexpression of Gadd34 enhances EBNA3C's ability to co-activate EBNA2 activation of the LMP1 promoter. Collectively the data suggest that EBNA3C interacts with Gadd34, activating the upstream component of the UPR (eIF2alpha phosphorylation) while preventing downstream UPR events (XBP1 activation and ATF6 cleavage).

Figure 1

Gadd34 aa 483-610 are required for EBNA3C association. a) 293-T cells were seeded in 6-well plates resulting in 70% confluence by day 2, and transfected with 0.5 μg of either psg5-EBNA3C alone (lane 1), psg5-Flag-Gadd34 (full-length, aa1-674, lane 2), or both EBNA3C and Flag-Gadd34 (1-674) (lane 3). In lanes 4-7 a combination of EBNA3C and the indicated Gadd34 mutants were transfected. Total DNA was 1.0 μg for each transfection. 18 hours post-transfection, cells were lysed in isotonic buffer containing 0.5% NP-40. Gadd34 or Gadd34 mutant proteins were immune precipitated with anti-flag affinity beads (M2-agarose), and precipitating proteins western blotted with α-EBNA3C antibody (A10) (top panel) or Flag antibody (bottom panel). In lane 7, 3% of starting lysates prior to immune precipitation (input) from the well transfected with psg5-EBNA3C alone (top panel), or psg5-Flag-Gadd34 alone (bottom panel) is shown.

Figure 2

EBNA3C interacts with Gadd34 by co-IP and co-purifies with Gadd34 by microcystin pulldown. a) IB4 cells (5 million for each treatment) were collected, lysed in isotonic 0.5% NP-40 buffer, and immune precipitations (IP) performed with either protein G alone (PG) or protein G with the addition of anti-EBNA3C (A10) sera (PG/anti-EBNA3C) Proteins were western blotted for Gadd34 (SC-H193), following IP. b) Burkitt's lymphoma BJAB cells or BJAB-EBNA3C cells (5 million for each treatment) were collected, lysed in isotonic 0.5% NP-40 buffer, containing additional 0.5% BSA, and incubated with the indicated sepharose beads conjugated to either protein G or microcystin LR. Lysates and beads were rotated for 1 hr at 4 degrees, extensively washed with PBS, and affinity purified proteins western blotted for the presence of EBNA3C (E3C) and Gadd34.

Figure 3

A: Gadd34 co-activation with EBNA3C requires aa483-610, and is independent of PP1 recruitment. A) BJAB cells (1 × 10⁷) were transfected with 5 μg of (-512/+72) LMP1p-Luc reporter construct, and 2 μg of psg5-EBNA2 (all lanes). psg5-EBNA3C (5 μg) was transfected(lanes 2-7). Gadd34 expression constructs were transfected as indicated (lanes 3-7) Luciferase values were normalized over beta-galactosidase levels obtained with co-transfection of 5 μg pgk-β-Gal plasmid. Reporter activation observed with EBNA2 alone is normalized to 1 (lane 1). Values are averages of duplicate observations in each experiment (repeated 3 times) plus standard error. A representative experiment is shown. B: Gadd34 180-483 is dominant negative in a low-dose EBNA3C co-activation assay. Reporter assay in BJAB cells with the indicated quantities of expression plasmids, as in figure 3a except that 100 ng (versus 5 μg) of psg5-EBNA3C expression plasmid was used. Each Gadd34 plasmids was titrated to determine stochiometry effects on EBNA3C, transfected at 0.1, 1.0 or 10.0 μg of DNA. C: Gadd34 does not co-activate transcription with EBNA2 in the absence of EBNA3C Reporter assay in BJAB cells with the indicated quantities of expression plasmids as in figure 3b. A representative experiment is shown. Protein expression levels of flag-tagged Gadd34, EBNA2 and EBNA3C are shown below.

Figure 4

A: EBNA3C effects on phosphorylation the translational control protein eIF2α. LCL 19-9 was maintained in the presence (+HT) or absence (-HT) of hydroxytamoxifen for 10 days. Shown are western blots from whole-cell lysates against EBNA3C (top row), serine 51 phosphorylated eIF2α (second row), total eIF2α (third row) and β-actin loading control (fourth row). By day 10, in the absence of HT, LCL19-9 cells had stopped dividing [7]. B: EBNA3C protects LCLs from activating the unfolded protein response. As in Figure 4a except that cells were also treated with either DMSO "C" or 0.5 μM Thapsigargin "Tg", for the 4 hrs preceding harvest. XBP1(s) and XBP1(u) were individually detected using isoform specific antibodies. An ATF6 antibody that detects only full length, uncleaved ATF6 (90 KDa) was used (Imgenex, San Diego, CA).