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Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells
Biochemical and Biophysical Research Communications 384 (2009) 301–305
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells q Wen T. Ji a, Lai Wang b, Ru C. Lin b, Wei R. Huang b, Hung J. Liu a,b,* a b
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan
a r t i c l e
i n f o
Article history: Received 13 April 2009 Available online 4 May 2009
Keywords: Avian reovirus eEF2 eIF4E eIF-4G
a b s t r a c t Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with avian reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Belonging to Reoviridae, avian reovirus (ARV) is an important pathogen in poultry. Sharing much similarity with mammalian reovirus (MRV), the virion particles of ARV possess two layers of capsid and ten genome segments of double-stranded RNAs [1]. The genome segments are divided into three size classes, designated as L (large), M (medium), and S (small), depending on their electrophoretic mobility that encode for at least 8 structural and 4 nonstructural proteins [2,3]. In the S-class segments of ARV, segment S1 contains three open reading frames which are translated into p10, p17, and rC proteins. Protein rC, encoded by the third open reading frame of S1 segment, is not only a cell attachment protein [1] but also an apoptosis inducer [4]. The nonstructural protein p17 is related to cell cycle retardation [5]. Avian reoviruses differ from their mammalian counterparts on their ability to cause massive cell fusion which is attributed to the protein p10 [6,7].
q The authors gratefully acknowledge financial support from the National Science Council (NSC 96-2313-B-020-001 and NSC 95-2313-B-020-009-MY3), Taiwan. * Corresponding author. Address: Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan. Fax: +886 8 7700447. E-mail address: [email protected] (H.J. Liu).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.04.116
Cap-dependent translation is initiated through assembly of a series of proteins called translation initiation factors [8]. It has been well documented that viral infection usually influences cellular protein synthesis. By suppressing the roles of various translation initiation factors, several viruses impede cap-dependent translation which potentially facilitates translation of viral transcripts or slows down production of cellular proteins related to innate defense [9]. Viruses such as poliovirus and encephalomyocarditis virus (EMCV) promote dephosphorylation of eIF4E [10]. A few viruses like human immunodeficiency virus (HIV) inhibit translation by degrading eIF4G [11]. In contrast, handicapping translation is also a mechanism by which cells actively respond to and restrict infection progress. Activated by double-stranded RNAs from the replication intermediate of RNA viruses, protein kinase R (PKR) phosphorylates eIF2a to halt production of viral proteins [12]. Unlike translation initiation factors, the function of eukaryotic translation elongation factor 2 (eEF2) is to catalyze the translocation of peptidyl-tRNA from the A site to the P site on the ribosome thereby facilitating elongation of peptide chains. The function of eEF2 is usually inhibited by eEF2 kinase (eEF2K)-mediated phosphorylation at Thr56 though eEF2 is also regulated by unknown mechanisms independent of eEF2K [13]. The activity of eEF2 is down-regulated by multiple upstream factors like Ca2+ signaling and AMP-activated protein kinase (AMPK). The mammalian target of rapamycin (mTOR) and MAPK p38 on the other hand are
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possible positive regulators of eEF2 [13–17]. The mTOR is an important downstream mediator of multiple signaling cascades including the PI3K-Akt and AMPK pathway. One critical function of mTOR is regulation of translation via controlling distinct factors such as 4E-BP1 that regulate the activity of a few translation initiation factors such as eIF4E. When phosphorylated by mTOR, 4EBP1 loses ability to sequester eIF4E hence allows initiation of cap-dependent translation. The protein p70S6K is also an important factor downstream of mTOR that plays similar roles in translation initiation control [18]. Modulation of the activity of various translation initiation factors is a critical strategy by which viruses regulate cellular protein production. Here, we provided the first evidences demonstrating that ARV-induced phosphorylation of eEF2 besides impeding the function of initiation factors through various mechanism which suggest that ARV infection influences host protein synthesis besides regulating cap-dependent translation.
added into each well. Cells were swirled gently for a few seconds and cultured continuously for 3 h. After incubation, medium was removed. Cells were washed twice with PBS. MTT metabolic product was resuspended in 500 ll DMSO. After gentle swirling for a few minutes, 50 ll supernatant from each well was transferred to optic plates and sent for detection. The optical density was evaluated at 570 nM, followed by subtracting background at 670 nM. Progeny titer. The progeny titer was determined using plaque assay. Twenty-four hours after particular treatment, cells and the culture medium were freeze–thawed three times to release virus particles. Serially diluted supernatant was used to infect fresh cells. After 1 h incubation, cells were washed twice with MEM to remove unabsorbed viruses and were further incubated in MEM for 1 h to allow viral penetration. To determine the titer of ARV, cells were maintained in fresh medium containing 20 mM NH4Cl to prevent secondary infection after washing again. The number of plaques was determined using an optical microscope about 24 h later.
Experimental procedures
Results and discussion
Cells and viruses. African green monkey kidney Vero cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 20 mM Hepes (pH 7.2). Cells were cultured in a 37 °C incubator supplied with 5% CO2. Vero cells were seeded in 4 well plates one day before each experiment. The experiment procedures were started after the cell confluence reached about 70–80%. Avian reovirus (ARV S1133) was propagated in Vero cells. When the cytopathic effects covered about 80% of the infected cells, the cells were frozen and thawed twice. Mixture was centrifuged at 3000g for 10 min to remove cellular debris. The supernatant was stored at 70 °C for future experiments. To prepare UV-irradiated ARV, the virus was exposed to short-wave UV light as previously described [19]. Antibodies and reagents. Dimethyl sulfoxide (DMSO), CGP57380, and thiazolyl blue tetrazolium bromide (MTT) were from Sigma (St. Louis, MO, USA). The PI3K inhibitor, Wortmannin, was from Merck (Frankfurter, Darmstadt, Germany). The mTOR inhibitor, rapamycin was purchased from A.G. Scientific, Inc. (San Diego, CA, USA). DMSO, the solvent used to dissolve above chemicals except for MTT was used as a negative control to treat cells. A monoclonal antibody against ARV rC protein was from our own laboratory stock [20]. All the other antibodies were from Cell Signaling Technology (Danvers, MA, USA). Isotope labeling. Vero cells mock or infected with ARV at indicated doses were labeled with [35S] methionine as previously described 24 h after particular treatment [21]. After labeling, cells were washed twice with PBS and lysed with 70 ll 2.5 Laemmli loading dye. Cells were harvested by scraping and boiled for 10 min. Equal amounts of samples were run on 10% SDS–PAGE gels. One panel of gels was transferred to PVDF membranes for detecting actin content. The label results were derived from exposing the dried gels to X-ray films (Kodak, Rochester, NY, USA). Electrophoresis and Western blot assay. Cells in 4 well plates were washed twice with PBS and lysed with 70 ll 2.5 Laemmli loading dye. Cells were harvested by scraping and boiled for 10 min. Equal amounts of samples were run on 10% SDS–PAGE gels and transferred to PVDF membranes. Expression of individual proteins was detected using respective antibodies, followed by the secondary antibody conjugated with horseradish peroxidase (HRP). After incubation with enhanced chemiluminescence (ECL plus) (Amersham Biosciences, Little Chalfont, Buckinghamshire, England), the membranes were exposed to X-ray films (Kodak, Rochester, NY, USA). Cell viability. Vero cells were seeded in 4 well plates one day before this procedure. At about 60% confluence cells were treated with different reagents. Twenty-four hours later 50 ll MTT was
Avian reovirus influences cellular translation initiation factors and elongation factor eEF2 Avian reovirus infection interfered with host cellular translation in a dose-dependant manner (Fig. 1A), similar to its mammalian counterpart which shuts off protein synthesis (22). Using pulsechase labeling, the translation status of cellular and viral protein was checked 24 h postinfection. In the result, the background which stands for the decrease in cellular proteins decreased with time. The level of newly synthesized viral proteins negatively correlated with that of cellular proteins (Fig. 1A, right panel), suggesting that ARV shut off cellular protein synthesis but not its own protein production. The protein translation status at different time points postinfection further confirmed that ARV gradually downregulated cellular protein synthesis (Fig. 1B). Accumulation of viral transcripts is one possible way through which viruses influence cellular protein productions since viral transcripts potentially compete with cellular ones for the translation machinery. To confirm whether the decreased production in host proteins was not simply due to accumulation of viral transcripts, the effect of ARV on translation initiation factors was checked. As shown, ARV notably reduced phosphorylation of eIF4G, eIF4E, 4EBP1, and p70S6K, all of them being factors involved in regulation of translation initiation (Fig. 2A). These results therefore suggested that ARV actively down-regulated host translation. Interestingly, infection with ARV obviously enhanced phosphorylation of a translation elongation factor, eEF2 (Fig. 2A). The phosphorylation of eEF2K was similarly down-regulated by ARV, which resulted in eEF2 phosphorylation hence inactivation. These results suggested ARV has the potential to reduce host protein production coordinately via disabling at least a translation elongation factor. The fact that ARV enhanced eEF2 phosphorylation in a time-dependent manner suggested that accumulation of viral products may be a major cause of eEF2 phosphorylation (Fig. 2B). Consistently, inactivation of ARV by UV did not cause eEF2 phosphorylation (Fig. 2C). Using 20 mM ammonium chloride (NH4Cl) to impede viral penetration also notably reduced viral protein level when using rC as a representative viral protein and phosphorylation of eEF2. These results confirmed that ARV replication enhances phosphorylation of eEF2. Avian reovirus survives inhibition of cap-dependent translation ARV infection obviously modulated the phosphorylation of several factors involved in translation initiation and elongation. In concern of the fact that accumulation of viral proteins was not
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0
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Fig. 1. Arrest of cellular protein synthesis by ARV. (A) Vero cells were infected with different doses of ARV. The status of protein synthesis 24 h after infection was determined using pulse-chase labeling. Cellular proteins were indicated by arrows as references. The relative level of translated proteins was shown after normalization against actin. The average level of cellular proteins was shown as black box when using each indicated proteins as 100%. The level of viral proteins was shown as open box using the proteins from the 10 MOI ARV-infected cells as 100%. Decrease of cellular products negatively correlated with ARV protein level. (B) The levels of cellular and viral proteins were also shown in different time points after inoculation of ARV at a dose of 10 PFU per cell. The figure was from triplicate results. Error bar represents standard deviation.
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Fig. 2. Enhancement of eEF2 phosphorylation by ARV. (A) Vero cells were infected with different doses of ARV. Cells were harvested for Western blot assay 24 h later. The level of phosphorylated eIF4G, eIF4E, 4E-BP1, and p70S6K notably decreased. On the other hand, phosphorylation of eEF2 obviously increased after infection. (B) The level of phosphorylated eEF2 increased with infection time in Vero cells inoculated with ARV at a dose of 10 PFU per cell. (C) Cells with different treatment were collected for Western blot assay 18 h later. Infection of cells with irradiated ARV caused no phosphorylation of eEF2. The negative effect of NH4Cl on ARV-induced eEF2 phosphorylation was also notable after adding 20 mM NH4Cl into the medium before viral inoculation.
W.T. Ji et al. / Biochemical and Biophysical Research Communications 384 (2009) 301–305
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Fig. 3. The effects of inhibitors against cap-dependent translation on ARV. (A) Vero cells mock or infected with ARV at a dose of 1 PFU per cell were treated with DMSO, 5 lM rapamycin, or 10 lM CGP57380. Cells were harvested for Western blot assay 24 h later. (B) Vero cells were treated with DMSO, 5 lM rapamycin, 10 lM CGP57380, or 1 lM Wortmannin. Cells were infected with ARV at the dose of 1 PFU per cell. The titer was determined 24 h later. Replication of ARV was not reduced by these inhibitors against cap-dependent translation. The figure was from triplicate results. Error bar represents standard deviation.
influenced by the decrease in cellular products, inhibitors of cellular cap-dependent translation were used here to confirm that viral replication was not negatively regulated by inhibiting cap-dependent translation. To exactly evaluate the influence of translation inhibitors on ARV propagation, cells were inoculated with a lower dose of ARV, 1 PFU per cell. Inhibition of mTOR by rapamycin slightly enhanced dephosphorylation of 4E-BP and increased the level of phosphorylated eIF4E (Fig. 3A). Inhibition of Mnk-1 by
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CGP57380 decreased the level of phosphorylated eIF4E in infected cells (Fig. 3A), caused no negative effect on the progeny titer of ARV (Fig. 3B). Compared to CGP57380, rapamycin even slightly raised ARV replication. Wortmannin, a PI3K inhibitor that had been successfully used to down-regulate cap-dependent translation in Vero cells [23], also did not down-regulate ARV multiplication. These results suggested that propagation of ARV continued during inhibition of cap-dependent translation.
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Fig. 4. The effect of rapamycin on viral translation. (A) Vero cells were treated with DMSO or 5 lM rapamycin. Cells were mock or infected with ARV at a dose 10 PFU per cell for 24 h. The level of phosphorylated eEF2 was slightly increased by 5 lM rapamycin in uninfected cells 24 h after treatment (left half). Rapamycin further raised eEF2 phosphorylation in ARV-infected cells (right half). The two halves were derived from the same film. (B) MTT analysis was used to evaluate the effect of rapamycin on cell numbers. Cell growth was negatively influenced by 5 lM rapamycin 24 h after treatment. (C) Viral syncytium as indicated by arrows in cells infected with ARV at the dose of 10 PFU per cell was not down-regulated by 5 lM rapamycin 24 h after inoculation. (D) The translation level of ARV was determined at the time point 24 h after treatment with 5 lM rapamycin or 10 lM CGP57380 using pulse-chase labeling. The average level of synthesized viral proteins in DMSO-treated cells was defined as 1. Translation of viral proteins was not negatively influenced by rapamycin. The figure was from triplicate results. Error bar represents standard deviation.
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References
eEF2 is regulated by multiple upstream signaling factors including mTOR [13]. As shown in Fig. 2A, ARV infection resulted in inhibition of mTOR as demonstrated by dephosphorylation of 4E-BP1 or p70S6K which might partially contribute to ARV-induced eEF2 phosphorylation. To evaluate the link between mTOR and eEF2 in Vero cells, uninfected cells were treated with rapamycin for 24 h. Compared to the results from the cells treated with DMSO, rapamycin slightly increased the level of phosphorylated eEF2 (Fig. 4A, left half). Interestingly, phosphorylation of eEF2 was synergistically enhanced by rapamycin in the cells infected with ARV at a dose of 10 PFU per cell (Fig. 4A, right half). These results suggested that down-regulation of mTOR activity by ARV partially facilitated eEF2 phosphorylation while the existence of other possible mechanisms could not be ruled out. Noteworthy is the fact that enhanced phosphorylation of eEF2 by rapamycin had no negative effects on ARV replication as indicated in Fig. 3. Although rapamycin reduced cell growth as evinced by MTT analysis (Fig. 4B), the scale of syncytium was slightly stronger in rapamycin-treated cells 24 h after infection (Fig. 4C). Further, the translation of ARV proteins 24 h after rapamycin treatment was not down-regulated (Fig. 4D). As shown, rapamycin even slightly enhanced translation of viral proteins. These results suggested that ARV survived eEF2 phosphorylation simultaneously induced by rapamycin and by the virus itself. Viruses have evolved various strategies to perturb cellular translation machinery for increasing translation compatibility or evading host defense [24,25]. However, phosphorylation of translation initiation factors is also a potential mechanism by which cells restrict viral infection though a few viruses have strategies that involve initiation factor phosphorylation [26]. Phosphorylation of eIF2a by PKR which is activated by dsRNA, a replication intermediate of RNA viruses, is a major mechanism by which host cell restricts viral infection. In general, most mammalian reoviruses not only induce but also benefit from eIF2a phosphorylation [22]. These reports increase the possibility that eEF2 phosphorylation may be beneficial or ARV replication. It is unclear why and how ARV infection induced eEF2 phosphorylation, although the obvious enhancement of eEF2 phosphorylation by rapamycin in infected cells was possibly due to increase in ARV replication. Rapamycin alone slightly raised eEF2 phosphorylation which may suggest that ARV probably down-regulated eEF2 function at least via inhibiting mTOR besides the fact that eEF2 is controlled by multiple mechanisms. Nevertheless, treatment with rapamycin did not reduce ARV replication, suggesting that eEF2 phosphorylation in viral infection is not deleterious for ARV. Compared to inhibition of eIF4E by CGP57380, rapamycin notably enhanced ARV translation. Phosphorylation of eEF2 may help maximize ARV multiplication during late infection times. How ARV withstands the negative effects of eEF2 phosphorylation remains an interesting issue to be solved. In conclusion, we provided here the first evidence that ARV-induced phosphorylation of eEF2 in Vero cells, highlighting the possibility that translation elongation factors may also play important roles in virus-induced translation arrest in infected cells.
[1] J. Benavente, J. Martinez-Costas, Avian reovirus: structure and biology, Virus Res. 123 (2007) 105–119. [2] G. Bodelon, L. Labrada, J. Martinez-Costas, J. Benavente, The avian reovirus genome segment S1 is a functionally tricistronic gene that expresses one structural and two nonstructural proteins in infected cells, Virology 290 (2001) 181–191. [3] R. Varela, J. Benavente, Protein coding assignment of avian reovirus strain S1133, J. Virol. 68 (1994) 6775–6777. [4] W.L. Shih, H.W. Hsu, M.H. Liao, L.H. Lee, H.J. Liu, Avian reovirus rC protein induces apoptosis in cultured cells, Virology 321 (2004) 65–74. [5] H.J. Liu, P.Y. Lin, J.W. Lee, H.Y. Hsu, W.L. Shih, Retardation of cell growth by avian reovirus p17 through the activation of p53 pathway, Biochem. Biophys. Res. Commun. 336 (2005) 709–715. [6] G. Bodelon, L. Labrada, J. Martinez-Costas, J. Benavente, Modification of late membrane permeability in avian reovirus-infected cells, J. Biol. Chem. 277 (2002) 17789–17796. [7] H.J. Liu, P.Y. Lin, L.R. Wang, H.Y. Hsu, M.H. Liao, W.L. Shih, Activation of small GTPases RhoA and Rac1 is required for avian reovirus p10-induced syncytium formation, Mol. Cells 26 (2008) 396–403. [8] W.C. Merrick, Cap-dependent and cap-independent translation in eukaryotic systems, Gene 332 (2004) 1–11. [9] R.J. Schneider, I. Mohr, Translation initiation and viral tricks, Trends Biochem. Sci. 28 (2003) 130–136. [10] M. Kleijn, C.L.J. Vrins, H.O. Voorma, A.A.M. Thomas, Phosphorylation state of the cap-binding protein eIF4E during viral infection, Virology 217 (1996) 486– 494. [11] C. Perales, L. Carrasco, I. Ventoso, Cleavage of eIF4G by HIV-1 protease: effects on translation, FEBS Lett. 533 (2003) 89–94. [12] J.O. Langland, J.M. Cameron, M.C. Heck, J.K. Jancovich, B.L. Jacobs, Inhibition of PKR by RNA and DNA viruses, Virus Res. 119 (2006) 100–110. [13] L.Q. Hong-Brown, C. Randell Brown, D.S. Huber, C.H. Lang, Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes, J. Biol. Chem. 282 (2007) 3702–3712. [14] G.J. Browne, C.G. Proud, Regulation of peptide-chain elongation in mammalian cells, Eur. J. Biochem. 269 (2002) 5360–5368. [15] G.J. Browne, C.G. Proud, A novel mTOR-regulated phosphorylation site in elongation factor 2 modulates the activity of the kinase and its binding to calmodulin, Mol. Cell. Biol. 24 (2004) 2986–2997. [16] A. Knebel, N. Morrice, P. Cohen, A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38d, EMBO J. 20 (2001) 4360–4369. [17] L. Wang, C.G. Proud, Regulation of the phosphorylation of elongation factor 2 by MEK-dependent signaling in adult rat cardiomyocytes, FEBS Lett. 531 (2002) 285–289. [18] A.R. Tee, J. Blenis, MTOR, translational control and human disease, Semin. Cell Dev. Biol. 16 (2005) 29–37. [19] L. Labrada, G. Bodelon, J. Vinuela, J. Benavente, Avian reoviruses cause apoptosis in cultured cells: viral uncoating, but not viral gene expression, is required for apoptosis induction, J. Virol. 76 (2002) 7932–7941. [20] J. Hsu, C.Y. Wang, L.H. Lee, W.L. Shih, C.I. Chang, H.L. Cheng, J.L.C. Chulu, W.T. Ji, H.J. Liu, Development and characterization of monoclonal antibodies against reovirus sigma C protein and their application in detection of reovirus isolates, Avian Pathol. 35 (2006) 320–326. [21] J.H. Connor, D.S. Lyles, Inhibition of host and viral translation during vesicular stomatitis virus infection, J. Biol. Chem. 280 (2005) 13512–13519. [22] J.A. Smith, S.C. Schmechel, A. Raghavan, M. Abelson, C. Reilly, M.G. Katze, R.J. Kaufman, P.R. Bohjanen, L.A. Schiff, Reovirus induces and benefits from an integrated cellular stress response, J. Virol. 80 (2006) 2019–2033. [23] D. Edgil, C. Polacek, E. Harris, Dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited, J. Virol. 80 (2006) 2976–2986. [24] M. Lopez-Lastra, A. Rivas, M.I. Barria, Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation, Biol. Res. 38 (2005) 121–146. [25] D.S. Lyles, Cytopathogenesis and inhibition of host gene expression by RNA viruses, Microbiol. Mol. Biol. Rev. 64 (2000) 709–724. [26] I. Ventoso, M.A. Sanz, S. Molina, J.J. Berlanga, L. Carrasco, M. Esteban, Translational resistance of late alphavirus mRNA to eIF2a phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR, Genes Dev. 20 (2006) 87–100.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Avian reovirus influences phosphorylation of several factors involved in host protein translation including eukaryotic translation elongation factor 2 (eEF2) in Vero cells q Wen T. Ji a, Lai Wang b, Ru C. Lin b, Wei R. Huang b, Hung J. Liu a,b,* a b
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan Graduate Institute of Biotechnology, National Pingtung University of Science and Technology, Pingtung 912, Taiwan
a r t i c l e
i n f o
Article history: Received 13 April 2009 Available online 4 May 2009
Keywords: Avian reovirus eEF2 eIF4E eIF-4G
a b s t r a c t Viral infection usually influences cellular protein synthesis either actively or passively via modification of various translation initiation factors. Here we demonstrated that infection with avian reovirus (ARV) interfered with cellular protein synthesis. This study demonstrated for the first time that ARV influenced the phosphorylation of translation initiation factors including eIF4E and eIF-4G. Interestingly, ARV also induced phosphorylation of eukaryotic translation elongation factor (eEF2) in a time- and dose-dependent manner. Inhibition of mTOR by rapamycin notably increased the level of phosphorylated eEF2 in infected cells. However, rapamycin did not show any negative effects on ARV replication, suggesting that phosphorylation of eEF2 in infected cells did not reduce ARV propagation. These results demonstrated for the first time that ARV promotes phosphorylation of eEF2 which in turn influenced host protein production not simply by modulating the function of translation initiation factors but also by regulating elongation factor eEF2. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Belonging to Reoviridae, avian reovirus (ARV) is an important pathogen in poultry. Sharing much similarity with mammalian reovirus (MRV), the virion particles of ARV possess two layers of capsid and ten genome segments of double-stranded RNAs [1]. The genome segments are divided into three size classes, designated as L (large), M (medium), and S (small), depending on their electrophoretic mobility that encode for at least 8 structural and 4 nonstructural proteins [2,3]. In the S-class segments of ARV, segment S1 contains three open reading frames which are translated into p10, p17, and rC proteins. Protein rC, encoded by the third open reading frame of S1 segment, is not only a cell attachment protein [1] but also an apoptosis inducer [4]. The nonstructural protein p17 is related to cell cycle retardation [5]. Avian reoviruses differ from their mammalian counterparts on their ability to cause massive cell fusion which is attributed to the protein p10 [6,7].
q The authors gratefully acknowledge financial support from the National Science Council (NSC 96-2313-B-020-001 and NSC 95-2313-B-020-009-MY3), Taiwan. * Corresponding author. Address: Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 912, Taiwan. Fax: +886 8 7700447. E-mail address: [email protected] (H.J. Liu).
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.04.116
Cap-dependent translation is initiated through assembly of a series of proteins called translation initiation factors [8]. It has been well documented that viral infection usually influences cellular protein synthesis. By suppressing the roles of various translation initiation factors, several viruses impede cap-dependent translation which potentially facilitates translation of viral transcripts or slows down production of cellular proteins related to innate defense [9]. Viruses such as poliovirus and encephalomyocarditis virus (EMCV) promote dephosphorylation of eIF4E [10]. A few viruses like human immunodeficiency virus (HIV) inhibit translation by degrading eIF4G [11]. In contrast, handicapping translation is also a mechanism by which cells actively respond to and restrict infection progress. Activated by double-stranded RNAs from the replication intermediate of RNA viruses, protein kinase R (PKR) phosphorylates eIF2a to halt production of viral proteins [12]. Unlike translation initiation factors, the function of eukaryotic translation elongation factor 2 (eEF2) is to catalyze the translocation of peptidyl-tRNA from the A site to the P site on the ribosome thereby facilitating elongation of peptide chains. The function of eEF2 is usually inhibited by eEF2 kinase (eEF2K)-mediated phosphorylation at Thr56 though eEF2 is also regulated by unknown mechanisms independent of eEF2K [13]. The activity of eEF2 is down-regulated by multiple upstream factors like Ca2+ signaling and AMP-activated protein kinase (AMPK). The mammalian target of rapamycin (mTOR) and MAPK p38 on the other hand are
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possible positive regulators of eEF2 [13–17]. The mTOR is an important downstream mediator of multiple signaling cascades including the PI3K-Akt and AMPK pathway. One critical function of mTOR is regulation of translation via controlling distinct factors such as 4E-BP1 that regulate the activity of a few translation initiation factors such as eIF4E. When phosphorylated by mTOR, 4EBP1 loses ability to sequester eIF4E hence allows initiation of cap-dependent translation. The protein p70S6K is also an important factor downstream of mTOR that plays similar roles in translation initiation control [18]. Modulation of the activity of various translation initiation factors is a critical strategy by which viruses regulate cellular protein production. Here, we provided the first evidences demonstrating that ARV-induced phosphorylation of eEF2 besides impeding the function of initiation factors through various mechanism which suggest that ARV infection influences host protein synthesis besides regulating cap-dependent translation.
added into each well. Cells were swirled gently for a few seconds and cultured continuously for 3 h. After incubation, medium was removed. Cells were washed twice with PBS. MTT metabolic product was resuspended in 500 ll DMSO. After gentle swirling for a few minutes, 50 ll supernatant from each well was transferred to optic plates and sent for detection. The optical density was evaluated at 570 nM, followed by subtracting background at 670 nM. Progeny titer. The progeny titer was determined using plaque assay. Twenty-four hours after particular treatment, cells and the culture medium were freeze–thawed three times to release virus particles. Serially diluted supernatant was used to infect fresh cells. After 1 h incubation, cells were washed twice with MEM to remove unabsorbed viruses and were further incubated in MEM for 1 h to allow viral penetration. To determine the titer of ARV, cells were maintained in fresh medium containing 20 mM NH4Cl to prevent secondary infection after washing again. The number of plaques was determined using an optical microscope about 24 h later.
Experimental procedures
Results and discussion
Cells and viruses. African green monkey kidney Vero cells were maintained in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 20 mM Hepes (pH 7.2). Cells were cultured in a 37 °C incubator supplied with 5% CO2. Vero cells were seeded in 4 well plates one day before each experiment. The experiment procedures were started after the cell confluence reached about 70–80%. Avian reovirus (ARV S1133) was propagated in Vero cells. When the cytopathic effects covered about 80% of the infected cells, the cells were frozen and thawed twice. Mixture was centrifuged at 3000g for 10 min to remove cellular debris. The supernatant was stored at 70 °C for future experiments. To prepare UV-irradiated ARV, the virus was exposed to short-wave UV light as previously described [19]. Antibodies and reagents. Dimethyl sulfoxide (DMSO), CGP57380, and thiazolyl blue tetrazolium bromide (MTT) were from Sigma (St. Louis, MO, USA). The PI3K inhibitor, Wortmannin, was from Merck (Frankfurter, Darmstadt, Germany). The mTOR inhibitor, rapamycin was purchased from A.G. Scientific, Inc. (San Diego, CA, USA). DMSO, the solvent used to dissolve above chemicals except for MTT was used as a negative control to treat cells. A monoclonal antibody against ARV rC protein was from our own laboratory stock [20]. All the other antibodies were from Cell Signaling Technology (Danvers, MA, USA). Isotope labeling. Vero cells mock or infected with ARV at indicated doses were labeled with [35S] methionine as previously described 24 h after particular treatment [21]. After labeling, cells were washed twice with PBS and lysed with 70 ll 2.5 Laemmli loading dye. Cells were harvested by scraping and boiled for 10 min. Equal amounts of samples were run on 10% SDS–PAGE gels. One panel of gels was transferred to PVDF membranes for detecting actin content. The label results were derived from exposing the dried gels to X-ray films (Kodak, Rochester, NY, USA). Electrophoresis and Western blot assay. Cells in 4 well plates were washed twice with PBS and lysed with 70 ll 2.5 Laemmli loading dye. Cells were harvested by scraping and boiled for 10 min. Equal amounts of samples were run on 10% SDS–PAGE gels and transferred to PVDF membranes. Expression of individual proteins was detected using respective antibodies, followed by the secondary antibody conjugated with horseradish peroxidase (HRP). After incubation with enhanced chemiluminescence (ECL plus) (Amersham Biosciences, Little Chalfont, Buckinghamshire, England), the membranes were exposed to X-ray films (Kodak, Rochester, NY, USA). Cell viability. Vero cells were seeded in 4 well plates one day before this procedure. At about 60% confluence cells were treated with different reagents. Twenty-four hours later 50 ll MTT was
Avian reovirus influences cellular translation initiation factors and elongation factor eEF2 Avian reovirus infection interfered with host cellular translation in a dose-dependant manner (Fig. 1A), similar to its mammalian counterpart which shuts off protein synthesis (22). Using pulsechase labeling, the translation status of cellular and viral protein was checked 24 h postinfection. In the result, the background which stands for the decrease in cellular proteins decreased with time. The level of newly synthesized viral proteins negatively correlated with that of cellular proteins (Fig. 1A, right panel), suggesting that ARV shut off cellular protein synthesis but not its own protein production. The protein translation status at different time points postinfection further confirmed that ARV gradually downregulated cellular protein synthesis (Fig. 1B). Accumulation of viral transcripts is one possible way through which viruses influence cellular protein productions since viral transcripts potentially compete with cellular ones for the translation machinery. To confirm whether the decreased production in host proteins was not simply due to accumulation of viral transcripts, the effect of ARV on translation initiation factors was checked. As shown, ARV notably reduced phosphorylation of eIF4G, eIF4E, 4EBP1, and p70S6K, all of them being factors involved in regulation of translation initiation (Fig. 2A). These results therefore suggested that ARV actively down-regulated host translation. Interestingly, infection with ARV obviously enhanced phosphorylation of a translation elongation factor, eEF2 (Fig. 2A). The phosphorylation of eEF2K was similarly down-regulated by ARV, which resulted in eEF2 phosphorylation hence inactivation. These results suggested ARV has the potential to reduce host protein production coordinately via disabling at least a translation elongation factor. The fact that ARV enhanced eEF2 phosphorylation in a time-dependent manner suggested that accumulation of viral products may be a major cause of eEF2 phosphorylation (Fig. 2B). Consistently, inactivation of ARV by UV did not cause eEF2 phosphorylation (Fig. 2C). Using 20 mM ammonium chloride (NH4Cl) to impede viral penetration also notably reduced viral protein level when using rC as a representative viral protein and phosphorylation of eEF2. These results confirmed that ARV replication enhances phosphorylation of eEF2. Avian reovirus survives inhibition of cap-dependent translation ARV infection obviously modulated the phosphorylation of several factors involved in translation initiation and elongation. In concern of the fact that accumulation of viral proteins was not
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Fig. 1. Arrest of cellular protein synthesis by ARV. (A) Vero cells were infected with different doses of ARV. The status of protein synthesis 24 h after infection was determined using pulse-chase labeling. Cellular proteins were indicated by arrows as references. The relative level of translated proteins was shown after normalization against actin. The average level of cellular proteins was shown as black box when using each indicated proteins as 100%. The level of viral proteins was shown as open box using the proteins from the 10 MOI ARV-infected cells as 100%. Decrease of cellular products negatively correlated with ARV protein level. (B) The levels of cellular and viral proteins were also shown in different time points after inoculation of ARV at a dose of 10 PFU per cell. The figure was from triplicate results. Error bar represents standard deviation.
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Fig. 2. Enhancement of eEF2 phosphorylation by ARV. (A) Vero cells were infected with different doses of ARV. Cells were harvested for Western blot assay 24 h later. The level of phosphorylated eIF4G, eIF4E, 4E-BP1, and p70S6K notably decreased. On the other hand, phosphorylation of eEF2 obviously increased after infection. (B) The level of phosphorylated eEF2 increased with infection time in Vero cells inoculated with ARV at a dose of 10 PFU per cell. (C) Cells with different treatment were collected for Western blot assay 18 h later. Infection of cells with irradiated ARV caused no phosphorylation of eEF2. The negative effect of NH4Cl on ARV-induced eEF2 phosphorylation was also notable after adding 20 mM NH4Cl into the medium before viral inoculation.
W.T. Ji et al. / Biochemical and Biophysical Research Communications 384 (2009) 301–305
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Fig. 3. The effects of inhibitors against cap-dependent translation on ARV. (A) Vero cells mock or infected with ARV at a dose of 1 PFU per cell were treated with DMSO, 5 lM rapamycin, or 10 lM CGP57380. Cells were harvested for Western blot assay 24 h later. (B) Vero cells were treated with DMSO, 5 lM rapamycin, 10 lM CGP57380, or 1 lM Wortmannin. Cells were infected with ARV at the dose of 1 PFU per cell. The titer was determined 24 h later. Replication of ARV was not reduced by these inhibitors against cap-dependent translation. The figure was from triplicate results. Error bar represents standard deviation.
influenced by the decrease in cellular products, inhibitors of cellular cap-dependent translation were used here to confirm that viral replication was not negatively regulated by inhibiting cap-dependent translation. To exactly evaluate the influence of translation inhibitors on ARV propagation, cells were inoculated with a lower dose of ARV, 1 PFU per cell. Inhibition of mTOR by rapamycin slightly enhanced dephosphorylation of 4E-BP and increased the level of phosphorylated eIF4E (Fig. 3A). Inhibition of Mnk-1 by
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CGP57380 decreased the level of phosphorylated eIF4E in infected cells (Fig. 3A), caused no negative effect on the progeny titer of ARV (Fig. 3B). Compared to CGP57380, rapamycin even slightly raised ARV replication. Wortmannin, a PI3K inhibitor that had been successfully used to down-regulate cap-dependent translation in Vero cells [23], also did not down-regulate ARV multiplication. These results suggested that propagation of ARV continued during inhibition of cap-dependent translation.
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Fig. 4. The effect of rapamycin on viral translation. (A) Vero cells were treated with DMSO or 5 lM rapamycin. Cells were mock or infected with ARV at a dose 10 PFU per cell for 24 h. The level of phosphorylated eEF2 was slightly increased by 5 lM rapamycin in uninfected cells 24 h after treatment (left half). Rapamycin further raised eEF2 phosphorylation in ARV-infected cells (right half). The two halves were derived from the same film. (B) MTT analysis was used to evaluate the effect of rapamycin on cell numbers. Cell growth was negatively influenced by 5 lM rapamycin 24 h after treatment. (C) Viral syncytium as indicated by arrows in cells infected with ARV at the dose of 10 PFU per cell was not down-regulated by 5 lM rapamycin 24 h after inoculation. (D) The translation level of ARV was determined at the time point 24 h after treatment with 5 lM rapamycin or 10 lM CGP57380 using pulse-chase labeling. The average level of synthesized viral proteins in DMSO-treated cells was defined as 1. Translation of viral proteins was not negatively influenced by rapamycin. The figure was from triplicate results. Error bar represents standard deviation.
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References
eEF2 is regulated by multiple upstream signaling factors including mTOR [13]. As shown in Fig. 2A, ARV infection resulted in inhibition of mTOR as demonstrated by dephosphorylation of 4E-BP1 or p70S6K which might partially contribute to ARV-induced eEF2 phosphorylation. To evaluate the link between mTOR and eEF2 in Vero cells, uninfected cells were treated with rapamycin for 24 h. Compared to the results from the cells treated with DMSO, rapamycin slightly increased the level of phosphorylated eEF2 (Fig. 4A, left half). Interestingly, phosphorylation of eEF2 was synergistically enhanced by rapamycin in the cells infected with ARV at a dose of 10 PFU per cell (Fig. 4A, right half). These results suggested that down-regulation of mTOR activity by ARV partially facilitated eEF2 phosphorylation while the existence of other possible mechanisms could not be ruled out. Noteworthy is the fact that enhanced phosphorylation of eEF2 by rapamycin had no negative effects on ARV replication as indicated in Fig. 3. Although rapamycin reduced cell growth as evinced by MTT analysis (Fig. 4B), the scale of syncytium was slightly stronger in rapamycin-treated cells 24 h after infection (Fig. 4C). Further, the translation of ARV proteins 24 h after rapamycin treatment was not down-regulated (Fig. 4D). As shown, rapamycin even slightly enhanced translation of viral proteins. These results suggested that ARV survived eEF2 phosphorylation simultaneously induced by rapamycin and by the virus itself. Viruses have evolved various strategies to perturb cellular translation machinery for increasing translation compatibility or evading host defense [24,25]. However, phosphorylation of translation initiation factors is also a potential mechanism by which cells restrict viral infection though a few viruses have strategies that involve initiation factor phosphorylation [26]. Phosphorylation of eIF2a by PKR which is activated by dsRNA, a replication intermediate of RNA viruses, is a major mechanism by which host cell restricts viral infection. In general, most mammalian reoviruses not only induce but also benefit from eIF2a phosphorylation [22]. These reports increase the possibility that eEF2 phosphorylation may be beneficial or ARV replication. It is unclear why and how ARV infection induced eEF2 phosphorylation, although the obvious enhancement of eEF2 phosphorylation by rapamycin in infected cells was possibly due to increase in ARV replication. Rapamycin alone slightly raised eEF2 phosphorylation which may suggest that ARV probably down-regulated eEF2 function at least via inhibiting mTOR besides the fact that eEF2 is controlled by multiple mechanisms. Nevertheless, treatment with rapamycin did not reduce ARV replication, suggesting that eEF2 phosphorylation in viral infection is not deleterious for ARV. Compared to inhibition of eIF4E by CGP57380, rapamycin notably enhanced ARV translation. Phosphorylation of eEF2 may help maximize ARV multiplication during late infection times. How ARV withstands the negative effects of eEF2 phosphorylation remains an interesting issue to be solved. In conclusion, we provided here the first evidence that ARV-induced phosphorylation of eEF2 in Vero cells, highlighting the possibility that translation elongation factors may also play important roles in virus-induced translation arrest in infected cells.
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