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. 2017 Apr:504:107-113.
doi: 10.1016/j.virol.2017.02.002. Epub 2017 Feb 9.

Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

Claire Barber  1 , Chris Netherton  1 , Lynnette Goatley  1 , Alice Moon  2 , Steve Goodbourn  2 , Linda Dixon  3
Affiliations

Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

Claire Barber et al. Virology. 2017 Apr.

Abstract

The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α.

Keywords: African swine fever virus; CHOP; EIF2α; Protein translation; Unfolded protein response.

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Figures

Fig. 1
Fig. 1
Alignment of DP71L with domains from GADD34 and ICP34.5 and mutants of DP71L Panel A) Shows an alignment of the long and short forms of DP71L with the C terminal domain of ICP34.5 of HSV-1 and GADD34. Within the C terminal region of ICP34.5 residues 233–248 (green) have been identified as the eIF2α binding domain (Li et al., 2011), whilst the eIF2α binding motif described by Rojas et al. (2015) in GADD34 is shown in blue. The predicted PP1 binding motif is highlighted in red. Panel B) shows the sequences of mutants of DP71L generated in this work. Numbers denote positions within wild type short DP71L sequences that correspond to mutations made. Dashed lines indicate the sequence is not altered from the wild type sequence and gaps show sequences deleted.
Fig. 2
Fig. 2
The residues V16, F18and 52–67 are required for function of DP71L. Vero cells were transfected with plasmids expressing HA epitope tagged wild type (A) or mutant DP71L, panel B, V16E, F18L or lacking residues 52–67 (panel C). At 24 h post-transfection cells were stimulated with 20 µg/ml tunicamycin for 8 h to induce expression of CHOP. Cells were then fixed in 4% PFA, permeabilised and labelled with DAPI, anti-HA and anti-CHOP antibodies. Primary antibodies were visualised with appropriate secondary reagents conjugated to Alexa 488 or Alexa 568 respectively. Arrows point to the nuclei of transfected cells. Scale bars represent 20 µm.
Fig. 3
Fig. 3
Wild type, but not mutant DP71L causes dephosphorylate eIF2α A) Vero cells were mock-transfected or transfected with wild type or mutant DP71L as indicated on the figure. At 24 h post-transfection cells were stimulated with 20 μg/ml tunicamycin for 8 h and then lysed. 20 μg of total protein from lysates was resolved by SDS-PAGE and transferred to membranes prior to blotting with antibodies against the HA epitope tag, phosphorylated and total eIF2α, CHOP and γ tubulin. The positions of molecular mass markers are indicated to the left of the gel (in Kilo Daltons). B) a) The relative level of phosphorylated to total eIF2α was determined by densitometry analysis using ImageJ software, and normalised to the ratio observed in lane 1. b) The relative ratio of CHOP to total eIF2α was determined as above, and expressed relative to the ratio observed in lane 8. The mean ratio was calculated from three independent experiments.
Fig. 4
Fig. 4
DP71L mutant V16E, F18L does not co-precipitate with PP1 Vero cells were transfected with plasmids expressing wild type or mutant DP71L, 24 h post-transfection lysates were harvested and incubated overnight with the HA affinity matrix at 4 °C with rotation. Lysates were pelleted and washed three times prior to re-suspension in SDS-PAGE loading buffer. Samples were resolved by SDS-PAGE, transferred to membranes by Western blot and probed against the HA epitope tag and PP1.
Fig. 5
Fig. 5
The V16E; F18L form of DP71L is unable to interact with protein phosphatase isoforms Panel B. Yeast strain PJ69-4α was transformed with pairs of plasmids expressing the indicated DNA binding hybrid and the phosphatase (PPC1) isoform fused to the yeast GAL4 activation domain. Yeast containing both plasmids were selected on synthetic drop-out medium lacking leucine and tryptophan (“-LW”) and then streaked onto synthetic drop-out medium lacking leucine, tryptophan and histidine and containing 5mJM or 10 mM 3-aminotriazole (“-LWH +3AT”). Growth on the latter is indicative of protein-protein interaction.
Fig. 6
Fig. 6
Wild type, but not mutant, DP71L acts as a translation enhancer Vero cells were co-transfected with equal amounts of the bi-cistronic reporter plasmid pIRES FF luc/Ren luc and pcDNA3, wild type or mutant DP71L as indicated. 24 h post-transfection cells were lysed and reporter activity assessed using the Dual-Luciferase Reporter Assay kit (Promega). The firefly (A) or renilla (B) reporter activity of control cells transfected with pcDNA3 was set at 100% and wild type or mutant activity expressed as a percentage relative to pcDNA3. Experiments were performed in triplicate three times. Error bars represent the standard deviation. Statistical analysis was carried out in GraphPad Prism using a one way ANOVA with multiple comparisons test. Asterisks represent a significant difference in value between WT DP71L and the mutants tested (*= P value of <0.5, ****= P value of <0.0001).
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References

    1. Alonso C., Galindo I., Cuesta-Geijo M.A., Cabezas M., Hernaez B., Muñoz-Moreno R. African swine fever virus-cell interactions: from virus entry to cell survival. Virus Res. 2013;173(1):42–57. - PMC - PubMed
    1. Bernales S., McDonald K.L., Walter P. Autophagy counterbalances endoplasmic reticulum expansion during the unfolded protein response. Plos Biol. 2006;4:2311–2324. - PMC - PubMed
    1. Brush M.H., Weiser D.C., Shenolikar S. Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol. Cell. Biol. 2003;23:1292–1303. - PMC - PubMed
    1. Chakrabarti A., Chen A.W., Varner J.D. A review of the mammalian unfolded protein response. Biotechnol. Bioeng. 2011;108:2777–2793. - PMC - PubMed
    1. Deng J., Lu P.D., Zhang Y.H., Scheuner D., Kaufman R.J., Sonenberg N., Harding H.P., Ron D. Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2. Mol. Cell. Biol. 2004;24:10161–10168. - PMC - PubMed

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