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JOURNAL OF VIROLOGY Dec 2009 p 12462 12472 Vol 83 No 23 0022 538X 09 12 00 doi 10 1128 JVI 01546 09 Copyright 2009 American Society for Microbiology All Rights Reserved Inhibition of Protein Kinase R Activation and Upregulation of GADD34 Expression Play a Synergistic Role in Facilitating Coronavirus Replication by Maintaining De Novo Protein Synthesis in Virus Infected Cells H17188 Xiaoxing Wang 1 Ying Liao 2 Pei Ling Yap 1 Kim J Png 1 James P Tam 2 and Ding Xiang Liu 1 2 Institute of Molecular and Cell Biology Proteos 61 Biopolis Drive Singapore 138673 1 and School of Biological Sciences Nanyang Technological University 60 Nanyang Drive Singapore 637551 2 Singapore Received 26 July 2009 Accepted 15 September 2009 A diversity of strategies is evolved by RNA viruses to manipulate the host translation machinery in order to create an optimal environment for viral replication and progeny production One of the common viral targets is the H9251 subunit of eukaryotic initiation factor 2 eIF 2H9251 In this report we show that phosphorylation of eIF 2H9251 was severely suppressed in human and animal cells infected with the coronavirus infectious bronchitis virus IBV To understand whether this suppression is through inhibition of protein kinase R PKR the double stranded RNA dependent kinase that is one of the main kinases responsible for phosphorylation of eIF 2H9251 cells infected with IBV were analyzed by Western blotting The results showed that the level of phosphorylated PKR was greatly reduced in IBV infected cells Overexpression of IBV structural and non structural proteins nsp demonstrated that nsp2 is a weak PKR antagonist Furthermore GADD34 a component of the protein phosphatase 1 PP1 complex which dephosphorylates eIF 2H9251 was significantly induced in IBV infected cells Inhibition of the PP1 activity by okadaic acid and overexpression of GADD34 eIF 2H9251 and PKR as well as their mutant constructs in virus infected cells showed that these viral regulatory strategies played a synergistic role in facilitating coronavirus replication Taken together these results confirm that IBV has developed a combination of two mechanisms i e blocking PKR activation and inducing GADD34 expression to maintain de novo protein synthesis in IBV infected cells and meanwhile to enhance viral replication Translation is a prime step for gene regulation in eukaryotic cells especially when cells are stressed by various signals in cluding viral infection Protein synthesis is globally limited in virus infected cells in many cases by alteration of the phos phorylation status of the H9251 subunit of eukaryotic initiation factor 2 eIF 2H9251 at serine 51 14 Phosphorylated eIF 2H9251 has a higher affinity for eIF 2B a guanine nucleotide exchange factor than the nonphosphorylated form and binds to it tightly Thus eIF 2H9251 is trapped and the formation of the 43S complex is impaired resulting in the inhibition of global translation Four kinases namely the heme regulated inhibitor 7 the double stranded RNA dsRNA activated protein kinase R PKR 54 the homologue of Saccharomyces cerevisiae pro tein kinase GCN2 19 and the PKR like endoplasmic reticu lum eIF 2H9251 kinase PERK 45 are known to phosphorylate eIF 2H9251 under different circumstances 46 These kinases re spond to different signals and play distinct roles in cells 9 Among the kinase family PKR is unique as it is activated by dsRNA often a by product during virus replication especially in cells infected with many RNA viruses 47 Therefore PKR plays a critical defensive role in viral infection 44 PKR is expressed at a low level but is markedly induced at the tran scriptional level by type I interferon alpha beta interferon 15 34 Binding to dsRNA induces autophosphorylation and dimerization of PKR leading to the activation of the kinase 37 Following activation PKR phosphorylates eIF 2H9251 result ing in the cessation of protein synthesis Because of the dele terious effects of this cellular response on viral replication many viruses have evolved mechanisms to countermeasure it 47 These mechanisms include a range of virus encoded RNA protein molecules directly interacting with PKR to block its activity 13 21 49 53 and proteins binding to and seques tering dsRNA 16 17 Some viruses can also inhibit the PKR activity by activating cellular inhibitors of PKR or cellular phosphatases PPs that dephosphorylate eIF 2H9251 5 33 In addition a recent report showed that translation of the late mRNA of alphaviruses was resistant to eIF 2H9251 phosphoryla tion as a highly stable RNA hairpin loop could bypass the requirement for a functional eIF 2H9251 51 Dephosphorylation of eIF 2H9251 is mediated by one of the major cellular protein PPs PP1 1 52 PP1 regulates a num ber of cellular functions through interactions of the catalytic subunit PP1c with more than 50 known or putative regulatory partners 8 Most of these interacting proteins target PP1c to specific subcellular locations to carry out its enzymatic reac tions One well established example is GADD34 a homologue of the mouse protein MyD116 GADD34 physically interacts with PP1c leading to enhanced dephosphorylation of eIF 2H9251 in vitro and in vivo 4 42 43 Induction of GADD34 by DNA Corresponding author Mailing address Institute of Molecular and Cell Biology Proteos 61 Biopolis Drive Singapore 138673 Sin gapore Phone 65 65869581 Fax 65 67791117 E mail dxliu imcb a star edu sg These authors contributed equally H17188 Published ahead of print on 23 September 2009 12462 on March 7 2015 by KUNGL TEKNISKA HOGSKOLAN http jvi asm org Downloaded from damage signals is well studied More recently its role in host virus interaction has been emerging For example Kazemi et al 18 reported that the E6 oncoprotein of papillomavirus type 18 associates with the GADD34 PP1 complex and medi ates translational recovery Vesicular stomatitis virus infection could induce GADD34 expression which in turn suppresses viral replication in mouse embryo fibroblasts by dephosphory lation of TSC2 in the mTOR pathway 36 Here we report that infection of human and animal cell lines with infectious bronchitis virus IBV a group 3 corona virus suppresses eIF 2H9251 phosphorylation and PKR activation and induces GADD34 expression Inhibition of PP1 activity by okadaic acid OA and manipulation of the expression activa tion of eIF 2H9251 PKR and GADD34 in virus infected cells ei ther by knockdown with small interfering RNA or by overex pression of wild type and mutant constructs showed that these viral regulatory strategies may play a synergistic role in facili tating coronavirus replication by maintaining de novo protein synthesis MATERIALS AND METHODS Cells viruses antibodies and reagents Vero and HUH7 cells were main tained in Dulbecco s modified Eagle s medium supplied with 10 fetal bovine serum FBS and 1 penicillin streptomycin Gibco BRL H1299 cells and H1299 cells stably expressing Renilla luciferase H1299 RL were maintained in RPMI medium with 10 FBS and 1 penicillin streptomycin All cells were grown at 37 C and supplied with 5 CO 2 The medium for cells was replaced with FBS free medium before and during virus infection The H1299 RL stable cell line was made by overexpressing the Renilla luciferase gene in the pXJ41neo vector and the positive cells were selected by use of G418 Sigma Aldrich A recombinant virus IBV Luc was constructed by replacing open reading frame ORF 3a 3b with the firefly luciferase gene by using an in vitro ligation protocol 48 The virus was recovered from Vero cells electroporated with in vitro transcripts generated from the full length IBV Luc cDNA All virus stocks were made in Vero cells by three repeated freeze thaw cycles and kept at H1100280 C UV inactivation of the virus was performed by exposing IBV to shortwave UV radiation of 120 000 mJ cm 2 at 254 nm of wavelength for 1 h within a CL 1000 cross linker UVP Polyclonal antibody against full length IBV M and N proteins were raised in rabbits Anti H9252 tubulin was purchased from Sigma Aldrich Anti eIF 2H9251 and anti phospho eIF 2H9251 anti p eIF 2H9251 at Ser51 were from Cell Signaling Anti phospho PKR anti p PKR at Thr451 anti PKR and anti GADD34 were from Abcam Anti H9252 actin antibody was from Santa Cruz Goat anti rabbit horseradish peroxidase linked secondary antibody and goat anti mouse horseradish peroxi dase linked secondary antibody were from Dako OA from Sigma Aldrich was dissolved in dimethyl sulfoxide DMSO For posttreatment cells were infected by virus and incubated at 37 C for 1 h before the addition of OA at the indicated concentrations Cells were then incubated for another 16 h before lysis Lipofectamine 2000 was obtained from Invitrogen Life Technologies DharmaFECT 2 was from Dharmacon Transient expression of plasmids in mammalian cells and establishment of the GADD34 knockdown cell line Cells grown to 80 confluence were trans fected with plasmid DNA by using Lipofectamine 2000 Invitrogen according to standard protocols Cells were either lysed at 18 to 24 h posttransfection for immunoblotting or infected with virus at a multiplicity of infection MOI of 1 for another 16 to 18 h before lysis Vero cells were stably transfected with a GADD34 short hairpin RNA con struct in the pSilencer 2 0 U6 vector Ambion Selection of positive clones was done in medium with G418 Immunoblotting Cells were washed with phosphate buffered saline PBS and scraped with a rubber policeman After centrifugation at 12 000 rpm for 1 min and removal of the supernatant pellets were lysed in lysis buffer 50 mM Tris Cl pH 7 4 150 mM NaCl 1 SDS 1 sodium deoxycholate supplemented with protease inhibitor Complete protease inhibitor cocktail from Roche The sam ples were boiled for 5 min prior to sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE The proteins were electrophoretically transferred to polyvinylidene difluoride membranes and immunoblotted according to the standard protocol with appropriate antibodies at proper dilutions The signals were detected using the ECL Plus chemiluminescence substrate kit GE Health care Densitometry The intensities of the bands were measured by a GS 710 cali brated imaging densitometer Bio Rad and analyzed using Molecular Analyst computer software Bio Rad 35 S metabolic labeling Metabolic labeling was carried out according to meth ods described previously 22 27 Vero cells were infected with IBV or mock infected for different time periods The medium was replaced with fresh Dul becco s modified Eagle s medium lacking methionine cysteine Gibco and cells were incubated at 37 C for 1 h Cells were then pulse labeled with 250 H9262Ci ml 35 S Met Cys labeling mixture from GE Healthcare for 2 h and then washed with PBS and lysed in SDS sample buffer Protein extracts were subjected to SDS PAGE and radioactive proteins were visualized by autoradiography RNA extraction and Northern blotting Cells were washed with PBS and lysed in TRIzol Fisher Scientific followed by the addition of 0 2 volumes of chloro form After centrifugation at 12 000 rpm at 4 C for 10 min the RNA fraction was transferred to a new tube and precipitated by 0 8 volumes of isopropanol After centrifugation RNA pellets were washed with 70 ethanol once and resus pended in RNase free H 2 O Northern blotting was carried out according to a standard protocol Roche Briefly RNA was separated on agarose gel and transferred to a positively charged nylon membrane After prehybridization a digoxigenin labeled DNA probe was added after denaturation at 100 C for 5 min and immediate cooling on ice The membrane was then probed with anti digoxi genin antibody Roche and signals were detected using the CDP star chemi luminescence substrate Roche Luciferase reporter assay Cells of 60 to 80 confluence grown on a 12 well plate were transfected with pXJ40F and pXJ40F GADD34 plasmid DNAs by Effectene according to the manufacturer s instructions Qiagen At 12 h post transfection the medium was replaced with serum free medium Cells were infected with IBV Luc at an MOI of 1 and were harvested for a luciferase reporter assay at the indicated time points 0 to 16 h postinfection The lucif erase reporter assay was performed according to manufacturer s manual Pro mega Luminescence was measured with a TD 20 20 luminometer Turner Designs H1299 cells of 90 confluence were transfected with pXJ40F vector pXJ40F PKR pXJ40F K296P pXJ40F eIF2H9251 pXJ40F S51D or pXJ40F S51A by use of Lipofectamine 2000 Invitrogen according to a standard protocol Cells were then infected with IBV Luc at an MOI of approximately 1 at 18 h posttransfec tion Cells were lysed in 1H11003 passive lysis buffer PLB Promega at 18 h postin fection h p i and the luciferase assay was performed according to the manu facturer s manual Promega H1299 RL cells seeded in a 12 well plate with 90 to 100 confluence were cotransfected with 0 5 H9262g reporter construct pcDNA FL and 1 H9262g pXJ40F PKR plus either 2 H9262g of plasmid encoding individual IBV proteins or 1 H9262g pXJ40F NS1 At 18 h posttransfection cells were lysed in 1H11003 PLB and subjected to luciferase assay according to the manufacturer s protocol Pro mega Plasmid construction Full length GADD34 was PCR amplified from cellular cDNAs and cloned into vector pXJ40F with a Flag tag under the control of a cytomegalovirus promoter Similarly full length PKR and eIF 2H9251 were PCR amplified from cellular cDNAs and cloned into pXJ40F generating pXJ40F PKR and pXJ40F eIF 2H9251 respectively PKR K296P and eIF 2H9251 S51A and S51D mutants were constructed by site directed mutagenesis Stratagene All IBV ORFs and nonstructural proteins nsps were obtained by reverse transcription PCR from virus infected cells by using appropriate primers and subsequently cloned into the pXJ40F vector RESULTS A relatively steady level of de novo host cell protein synthe sis was maintained in Vero cells infected with the coronavirus IBV The effect of IBV infection on host cell protein synthesis was analyzed in two ways First Vero cells infected with IBV at an MOI of approximately 1 were harvested at 0 8 16 and 24 h p i Total proteins were separated by SDS PAGE and visual ized by direct staining with Coomassie blue Similar levels of cellular protein synthesis were detected in mock and IBV infected Vero cells up to 24 h p i Fig 1 upper panel At 16 and 24 h p i a protein band with an apparent molecular mass of approximately 32 kDa was detected in IBV infected cells VOL 83 2009 REGULATION OF PROTEIN SYNTHESIS IN IBV INFECTED CELLS 12463 on March 7 2015 by KUNGL TEKNISKA HOGSKOLAN http jvi asm org Downloaded from Fig 1 upper panel lanes 7 and 9 It may represent the IBV membrane M protein These results suggest that IBV infec tion did not significantly affect the steady level of host proteins up to 24 h p i Metabolic labeling of IBV infected cells with 35 S Met Cys was then carried out to study the kinetics of de novo protein synthesis in IBV infected cells in a time course experiment The infected cells were labeled with 35 S Met Cys at 0 6 14 and 22 h p i for 2 h and harvested Cell lysates were prepared and total proteins were separated with SDS PAGE The re sults showed that de novo host cell protein synthesis was not significantly affected up to 8 h p i Fig 1 lower panel At 16 and 24 h p i two major proteins with molecular masses of approximately 32 and 46 kDa representing the major IBV structural proteins M and nucleocapsid N respectively were detected in IBV infected cells Fig 1 lower panel but not in mock infected cells Fig 1 lower panel lanes 6 and 8 It was noted that host cell protein translation was slightly reduced in both mock and IBV infected cells at late stages of the time course This was probably due to prolonged incubation of cells in serum free media Nevertheless the overall levels of protein synthesis in mock and IBV infected cells were comparable which is consistent with previous studies 23 24 31 38 These results demonstrate that de novo synthesis of host cell proteins is maintained in IBV infected cells even at late stages of the infection cycle although de novo synthesis of viral proteins becomes a more predominant event The expression and phosphorylation of eIF 2H9251 were down regulated in IBV infected Vero H1299 and HUH7 cells and IBV replication was inhibited by overexpression of the wild type and a phosphomimetic mutant of eIF 2H9251 It is well known that phosphorylation of eIF 2H9251 leads to the loss of functional eIF 2H9251 capable of binding to GTP and forming tertiary initia tion complex with initiator tRNA Met resulting in translation inhibition To understand the mechanisms that control de novo protein synthesis in IBV infected cells the expression and phosphorylation of eIF 2H9251 in IBV infected Vero cells were examined in detailed time course experiments Total cell ly sates were resolved with SDS PAGE and probed with an an tibody specific for p eIF 2H9251 at the serine 51 position and an anti eIF 2H9251 antibody It showed that in IBV infected Vero cells p eIF 2H9251 was dramatically decreased after 8 h p i Fig 2a lanes 4 to 8 The total eIF 2H9251 remained largely stable during IBV infection in Vero cells although a gradual de crease of the protein in this experiment and repeated experi ments was observed after normalization with the tubulin load ing control Fig 2a The relative levels of p eIF 2H9251 and eIF 2H9251 were determined by measuring the density of each band by densitometry and the calculated ratios of p eIF 2H9251 to total eIF 2H9251 are 0 64 0 60 0 04 0 03 0 03 0 02 and 0 01 at 4 8 12 16 20 24 and 28 h p i respectively Interestingly a specific band migrating slightly more rapidly than the eIF 2H9251 band was detected from 12 h p i onwards Fig 2a The identity of this band is currently unknown It may represent a cleaved de graded form of eIF 2H9251 This observation combined with the low level of host proteins labeled at a late stage of virus infec tion suggested that protein degradation might take place at these time points For a control for the efficiency of IBV infection the same membrane was probed with anti IBV M antibodies indicating a gradual increase in virus infection over time Fig 2a A similar analysis was conducted with IBV infected HUH7 and H1299 cells revealing a similar profile of eIF 2H9251 expres sion and phosphorylation Levels of p eIF 2H9251 dropped signif icantly from 8 h p i onwards Fig 2a lanes 3 to 8 Subse quently it became barely detectable especially at 24 and 28 h p i Fig 2a lanes 7 and 8 The ratios of p eIF 2H9251 to total eIF 2H9251 in HUH7 cells are 0 28 0 40 0 11 0 04 0 03 0 03 0 02 and 0 003 at 0 4 8 12 16 20 24 and 28 h p i respectively The ratios for H1299 cells are 0 10 0 14 0 15 0 08 0 008 0 007 0 005 and 0 002 at 0 4 8 12 16 20 24 and 28 h p i respectively Once again detection of the IBV M protein was carried out for an indication of IBV infection in the two cell types showing gradually increased viral infection over time Fig 2a Due to the loading of a large amount of samples for protein analysis in this experiment and prolonged exposure time a relatively high level of p eIF 2H9251 was detected in both cell lines at 0 h p i In fact a high basal level of p eIF 2H9251 was also detected in other studies 12 41 To further verify the results a parallel experiment was con ducted with H1299 cells incubated with UV inactivated IBV As shown in Fig 2a the basal phosphorylation levels of eIF 2H9251 in cells infected with live IBV and UV inactivated IBV UV IBV at 0 h were similar In contrast to that in IBV infected cells the p eIF 2H9251 level in cells incubated with UV IBV was slightly elevated and then maintained at a stable level Fig 2a This slightly increased detection of p eIF 2H9251 may be due to serum starvation The absence of M protein expression in cells incubated with UV IBV confirmed total inactivation of the virus Fig 2a To investigate the role of eIF 2H9251 phosphorylation on IBV infection three constructs