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      EBNA3C interacts with Gadd34 and counteracts the unfolded protein response

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      Virology Journal
      BioMed Central

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          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 ICPγ34.5 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 interaction[ 1]. 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 eIF2α 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 (eIF2α phosphorylation) while preventing downstream UPR events (XBP1 activation and ATF6 cleavage).

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          The gamma(1)34.5 protein of herpes simplex virus 1 complexes with protein phosphatase 1alpha to dephosphorylate the alpha subunit of the eukaryotic translation initiation factor 2 and preclude the shutoff of protein synthesis by double-stranded RNA-activated protein kinase.

          In human cells infected with herpes simplex virus 1 the double-stranded RNA-dependent protein kinase (PKR) is activated but phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2) and total shutoff of protein synthesis is observed only in cells infected with gamma(1)z34.5- mutants. The carboxyl-terminal 64 aa of gamma(1)34.5 protein are homologous to the corresponding domain of MyD116, the murine growth arrest and DNA damage gene 34 (GADD34) protein and the two domains are functionally interchangeable in infected cells. This report shows that (i) the carboxyl terminus of MyD116 interacts with protein phosphatase 1alpha in yeast, and both MyD116 and gamma(1)34.5 interact with protein phosphatase 1alpha in vitro; (ii) protein synthesis in infected cells is strongly inhibited by okadaic acid, a phosphatase 1 inhibitor; and (iii) the alpha subunit in purified eIF-2 phosphorylated in vitro is specifically dephosphorylated by S10 fractions of wild-type infected cells at a rate 3000 times that of mock-infected cells, whereas the eIF-2alpha-P phosphatase activity of gamma(1)34.5- virus infected cells is lower than that of mock-infected cells. The eIF-2alpha-P phosphatase activities are sensitive to inhibitor 2. In contrast to eIF-2alpha-P phosphatase activity, extracts of mock-infected cells exhibit a 2-fold higher phosphatase activity on [32P]phosphorylase than extracts of infected cells. These results indicate that in infected cells, gamma(1)34.5 interacts with and redirects phosphatase to dephosphorylate eIF-2alpha to enable continued protein synthesis despite the presence of activated PKR. The GADD34 protein may have a similar function in eukaryotic cells. The proposed mechanism for maintenance of protein synthesis in the face of double-stranded RNA accumulation is different from that described for viruses examined to date.
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            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.

            The growth arrest and DNA damage-inducible protein, GADD34, associates with protein phosphatase 1 (PP1) and promotes in vitro dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2, (eIF-2 alpha). In this report, we show that the expression of human GADD34 in cultured cells reversed eIF-2 alpha phosphorylation induced by thapsigargin and tunicamycin, agents that promote protein unfolding in the endoplasmic reticulum (ER). GADD34 expression also reversed eIF-2 alpha phosphorylation induced by okadaic acid but not that induced by another phosphatase inhibitor, calyculin A (CA), which is a result consistent with PP1 being a component of the GADD34-assembled eIF-2 alpha phosphatase. Structure-function studies identified a bipartite C-terminal domain in GADD34 that encompassed a canonical PP1-binding motif, KVRF, and a novel RARA sequence, both of which were required for PP1 binding. N-terminal deletions of GADD34 established that while PP1 binding was necessary, it was not sufficient to promote eIF-2 alpha dephosphorylation in cells. Imaging of green fluorescent protein (GFP)-GADD34 proteins showed that the N-terminal 180 residues directed the localization of GADD34 at the ER and that GADD34 targeted the alpha isoform of PP1 to the ER. These data provide new insights into the mode of action of GADD34 in assembling an ER-associated eIF-2 alpha phosphatase that regulates protein translation in mammalian cells.
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              Coronavirus infection modulates the unfolded protein response and mediates sustained translational repression.

              During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6alpha. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing alpha-mannosidase-like protein, and p58(IPK), or expression of UPR reporter constructs. Translation initiation factor eIF2alpha is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2alpha during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed.
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                Author and article information

                Journal
                Virol J
                Virology Journal
                BioMed Central
                1743-422X
                2009
                29 December 2009
                : 6
                : 231
                Affiliations
                [1 ]University of Pittsburgh and Children's Hospital of Pittsburgh, One Children's Hospital Drive, 4401 Penn Ave, Pittsburgh, PA 15224, USA
                [2 ]Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo 060-0815, Japan
                Article
                1743-422X-6-231
                10.1186/1743-422X-6-231
                2805635
                20040105
                235fe4be-407b-4e17-90db-2c5cbd7dcd15
                Copyright ©2009 Garrido et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 October 2009
                : 29 December 2009
                Categories
                Research

                Microbiology & Virology
                Microbiology & Virology

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