Increased Susceptibility to DNA Virus Infection in Micewith a GCN2 Mutation

Sungyong Won,a Celine Eidenschenk,a* Carrie N. Arnold,a Owen M. Siggs,a Lei Sun,a* Katharina Brandl,a Tina-Marie Mullen,b

Glen R. Nemerow,b Eva Marie Y. Moresco,a and Bruce Beutlera*

Department of Geneticsa and Department of Immunology and Microbial Sciencesb, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California92037, USA

The downregulation of translation through eIF2� phosphorylation is a cellular response to diverse stresses, including viral in-fection, and is mediated by the GCN2 kinase, protein kinase R (PKR), protein kinase-like endoplasmic reticulum kinase (PERK),and heme-regulated inhibitor kinase (HRI). Although PKR plays a major role in defense against viruses, other eIF2� kinases alsomay respond to viral infection and contribute to the shutdown of protein synthesis. Here we describe the recessive, loss-of-function mutation atchoum (atc) in Eif2ak4, encoding GCN2, which increased susceptibility to infection by the double-strandedDNA viruses mouse cytomegalovirus (MCMV) and human adenovirus. This mutation was identified by screening macrophagesisolated from mice carrying N-ethyl-N-nitrosourea (ENU)-induced mutations. Cells from Eif2ak4atc/atc mice failed to phosphory-late eIF2� in response to MCMV. Importantly, homozygous Eif2ak4atc mice showed a modest increase in susceptibility toMCMV infection, demonstrating that translational arrest dependent on GCN2 contributes to the antiviral response in vivo.

The activation of innate immune sensors of viral infection, suchas the nucleic acid-sensing Toll-like receptors and RIG-I-like

receptors, induces the production of inflammatory cytokines andtype I interferons (IFN). IFNs induce an antiviral state that de-pends on the expression of genes driving, in addition to innate andadaptive immune responses per se, cellular activities that promotean environment detrimental to virus survival and proliferation.For example, viruses depend on the host translational machineryto synthesize their proteins, and mammalian cells downregulatetranslation as one means of countering viral infection.

GCN2 kinase, protein kinase R (PKR), protein kinase-like en-doplasmic reticulum kinase (PERK), and heme-regulated inhibi-tor kinase (HRI) phosphorylate the eukaryotic translation initia-tion factor eIF2� at conserved serine residue 51 (40). Whenphosphorylated, eIF2� binds and functionally sequesters the gua-nine nucleotide exchange factor eIF2B, thereby decreasing levelsof GTP-bound eIF2 required for translation initiation (21, 32).Three of the four eIF2� kinases respond to distinct environmentalstresses: PERK to misfolded proteins in the endoplasmic reticu-lum (ER stress) (14, 39), HRI to heme deprivation and oxidativeand heat stresses in erythroid tissues (13, 23), and GCN2 to aminoacid deprivation, UV irradiation, and proteasome inhibition (9,19, 41, 44). The fourth eIF2� kinase, PKR, is induced by type I IFNand activated by double-stranded RNA (dsRNA) (10), which isderived from dsRNA viruses and synthesized as an intermediateduring the replication of single-stranded RNA viruses and dsDNAviruses. Thus, eIF2� phosphorylation by PKR leads to a globalblock of protein synthesis that in turn hinders viral protein pro-duction. The existence of an eIF2� kinase specialized to sense andrespond to viral infection underscores the effectiveness of thisantiviral strategy. Indeed, many viruses have evolved countermea-sures to PKR signaling (6, 15, 24, 29, 33, 37).

A few reports also have implicated eIF2� phosphorylation byPERK and GCN2 in antiviral responses. PERK is activated duringherpes simplex virus 1 infection, likely as a result of accumulatedviral proteins in the ER (7) and during vesicular stomatitis virusinfections through an unknown mechanism (3). GCN2 is directly

activated in vitro by the binding of the Sindbis virus genomic RNAto the histidyl-tRNA synthetase-like domain of GCN2, andGCN2-deficient mice infected with Sindbis virus display elevatedviral titers in the brain relative to those of wild-type mice (4).Whether GCN2 or PERK is involved in defense against other vi-ruses is not known.

We carried out a genetic screen to identify genes important forthe early control of DNA viruses, represented by mouse cytomeg-alovirus (MCMV) and human adenovirus, or RNA viruses, rep-resented by influenza. Using thioglycolate-elicited peritonealmacrophages from N-ethyl-N-nitrosourea (ENU)-mutagenizedmice, we identified a mutant strain, atchoum (atc), with macro-phages that became infected by MCMV and human adenoviruswith increased frequency relative to that of wild-type macro-phages. The phenotype was ascribed to a missense mutation of thegene encoding GCN2. The atc mutation abrogated the phosphor-ylation of eIF2� in response to MCMV infection. Moreover, themutation increased susceptibility to a sublethal inoculum ofMCMV in homozygous mice.

MATERIALS AND METHODSMice, cells, and viruses. The Eif2ak4atchoum, Ifnar1m1Btlr (macro-1;3822164), Ifnar2m1Btlr (macro-2; 3841008), Stat1m1Btlr (domino; 3619019),Tlr9m1Btlr (CpG1; 3038816), Unc93b13d (3619211), and Myd88poc (poco-curante; 3641255) alleles were generated on a C57BL/6J background byN-ethyl-N-nitrosourea (11) and are described at http://mutagenetix

Received 12 July 2011 Accepted 11 November 2011

Published ahead of print 23 November 2011

Address correspondence to B. Beutler, Bruce.Beutler@UTSouthwestern.edu.

* Present address: C. Eidenschenk, Department of Immunology, Genentech, Inc.,South San Francisco, California, USA; L. Sun and B. Beutler, Center for Genetics ofHost Defense, UT Southwestern Medical Center, Dallas, Texas, USA.

S.W. and C.E. contributed equally to this work.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.05660-11

1802 jvi.asm.org 0022-538X/12/$12.00 Journal of Virology p. 1802–1808

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

.utsouthwestern.edu/ (Mouse Genome Informatics database accessionsnumbers are indicated in parentheses). Tlr3�/� and Tlr7�/� mice werefrom Richard Flavell (Yale University, New Haven, CT) and Shizuo Akira(Osaka University, Osaka, Japan), respectively. Ifnar1�/� and Ifngr1�/�

mice were from Jonathan Sprent (Garvan Institute of Medical Research,Darlinghurst, New South Wales, Australia). C57BL/6J mice used for mu-tagenesis and C57BL/10J mice were from The Jackson Laboratory; allother C57BL/6J mice were from the breeding colony of The Scripps Re-search Institute (La Jolla, CA). All animals were housed in The ScrippsResearch Institute Animal Facility. All procedures using animals were inaccordance with guidelines of the Institutional Animal Care and UseCommittee.

Macrophages were induced in mice by the intraperitoneal (i.p.) injec-tion of 1.5 to 2 ml 4% (wt/vol) Brewer’s thioglycolate medium powder(BBL Microbiology Systems) in distilled water 4 days prior to isolation.Macrophages were collected by lavage, concentrated by centrifugation,and resuspended in PEC medium (5% [vol/vol] heat-inactivated fetalbovine serum [Atlanta Biologicals], 200 IU/ml penicillin, 200 mg/mlstreptomycin in HEPES-buffered saline). Mouse embryonic fibroblastswere prepared from day 13.5 embryos using a standard protocol.

MCMV-green fluorescent protein (GFP) (27) was a gift from ChrisBenedict (La Jolla Institute of Allergy and Immunology, La Jolla, CA). Itwas propagated on mouse embryonic fibroblasts and purified as describedpreviously (22). MCMV used for in vivo infections was prepared andmaintained as described elsewhere (http://mutagenetix.utsouthwestern.edu/protocol/protocol_rec.cfm?pid�5). The nonreplicating hAd5-F16-GFP is the human adenovirus 5 serotype expressing the fiber protein fromserotype 16 and tagged with GFP (16). hAd5-F16-GFP was a gift fromGlen Nemerow (The Scripps Research Institute) and was grown in 293cells and purified by CsCl gradient. The titer was determined as describedpreviously (16, 38). PR8 influenza A virus was prepared and maintained asdescribed previously (http://mutagenetix.utsouthwestern.edu/protocol/protocol_rec.cfm?pid�18).

Virus infections and MCMV quantitation. The infection of macro-phages and the measurement of infected cells was performed as describedpreviously (http://mutagenetix.utsouthwestern.edu/protocol/protocol_rec.cfm?pid�10). Briefly, viruses were added at the indicated dosages to105 macrophages per well in a 96-well plate and incubated at 37°C with 5%CO2 in a humidified incubator for 24 h (MCMV-GFP or PR8) or 72 h(hAd5-F16-GFP). Mice were infected with MCMV by the intraperitonealinjection of the indicated dose. The number of MCMV PFU in culturesupernatant of wild-type or Eif2ak4atc/atc fibroblasts was determined byplaque assay in NIH 3T3 cells as described previously (31).

dsDNA stimulation of macrophages and cytokine measurements.The stimulation of macrophages with dsDNA and the measurement oftype I IFN production was performed as described elsewhere (http://mutagenetix.utsouthwestern.edu/protocol/protocol_rec.cfm?pid�6).Briefly, 0.1 �g of dsDNA and 0.25 �l Lipofectamine 2000 (Invitrogen) ina total of 50 �l of Opti-MEM medium (Gibco) was added to 5 � 104

macrophages per well in a 96-well plate and incubated at 37°C with 5%CO2 in a humidified incubator for 16 h. Following incubation, the con-centration of type I IFN in the supernatant was assayed using aninterferon-stimulated response element (ISRE)-driven luciferase reporterbioassay. Type I IFN in the supernatant of hAd5-F16-GFP-infected mac-rophages was measured 72 h postinfection using the same protocol.

BSA, whole-genome sequencing, and genotyping. Bulk segregationanalysis (BSA) was performed as described previously (42) using F2 micegrouped into mutant and wild-type groups based on the percentage ofmacrophages infected by hAd5-F16-GFP. DNA for whole-genome se-quencing was prepared from two different atc homozygous mice as de-scribed previously (2), and SOLiD sequencing was performed using threeslides according to SOLiD 3 (2 slides) or SOLiD 4 (1 slide) instructionsprovided by the manufacturer (Applied Biosystems). SOLiD data wereanalyzed as described previously (2).

Mice were genotyped by sequencing PCR products, amplified from

genomic DNA, across the particular mutation site using a 3730xl se-quencer (Applied Biosystems). PCR primers were the following:Eif2ak4atc (forward, 5=-AATTGGCTGGGACGGTGTCAAG-3=; reverse,5=-GGAAGCACTTTAAATGCTCGCCAC-3=); Ifnar1macro-1 (forward,5=-AGAACAGCTTGCCACTTCACTGG-3=; reverse, 5=-GCAGAGAAGCCTTAGCCTTAGAAGAAC-3=); Ifnar2macro-2 (forward, 5=-TTGATACCACAGCGGAAGGTGAGC-3=; reverse, 5=-AACCATAGGCGGGACACATTAACTG-3=). Sequencing primers were the following: Eif2ak4atc

(forward, 5=-GCTGTAACTTAGTGTACAGGGAC-3=; reverse, 5=-CATCGGTAGTGAGCACTCTACAG-3=); Ifnar1macro-1 (forward, 5=-CCCAGGGTAGCTTCAAACTTATG-3=; reverse, 5=-TCACAAAGTTCCTGGGTAGC-3=); Ifnar2macro-2 (forward, 5=-CCTCTCTGCTTAGAGGACAGATG-3=; reverse, 5=-CAAGGCCGTTTCCTGAATATG-3=).

Reverse transcriptase PCR (RT-PCR). Total RNA was preparedfrom thioglycolate-elicited peritoneal macrophages using TRIzol (In-vitrogen). The RNA was reverse transcribed using a RETROscript first-strand synthesis kit (Ambion). Five products were amplified using thefollowing PCR primers: forward, 5=-GAGAGCTATTCGCAGCGACAGG-3=; reverse, 5=-TGTCAAGCTAGTGATTTCCAAACGTTCC-3=. ThePCR products were purified using a QIAquick PCR purification kit (Qia-gen) and sequenced using BigDye terminator v3.1 on a 3730xl sequencer(Applied Biosystems) using the following primers: forward, 5=-ATTTACGGCTCGGACTTCCAG-3=; reverse, 5=-CTCTGAATCTCGTGGAGGATTTC-3=.

Western blotting and antibodies. Macrophages or fibroblasts (105)were lysed in Laemmli sample buffer. Proteins were separated by SDS-PAGE and immunoblotted using the antibodies to GCN2 (3302; CellSignaling Technology), eIF2� (9722; Cell Signaling Technology), eIF2�(phospho-Ser51) (9721; Cell Signaling Technology), and �-actin (sc-47778; Santa Cruz Biotechnology).

Immunization and analysis of antibody responses. Age- and sex-matched mice were injected i.p. with 2 � 106 IU of a recombinant SemlikiForest virus (SFV) vector encoding the model antigen, �-galactosidase(rSFV-�Gal), in a total volume of 200 �l of sterile 0.9% saline. Ten dayslater, the mice were injected i.p. with 50 mg of NP28-AECM-Ficoll (Bio-search Technologies) in a total volume of 200 �l of sterile 0.9% saline. Onday 14 after immunization with rSFV-�Gal (and day 5 after immuniza-tion with NP-Ficoll), mice were anesthetized with isoflurane and bloodfrom the retro-orbital plexus of each animal was collected in serum sep-arator tubes (Becton Dickinson). For the detection of specific antibodies,polyvinyl chloride microtiter 96-well round-bottom plates (Costar) werecoated overnight at 4°C with 2 �g/ml �-galactosidase (Roche) or 5 �g/mlNP23-bovine serum albumin (Biosearch Technologies). Plates werewashed in Dulbecco’s phosphate-buffered saline (dPBS) without calciumand magnesium and blocked with 5% milk. Serum samples were seriallydiluted in 1% milk. Plates were incubated with horseradish peroxidase(HRP)-conjugated goat anti-mouse IgM or IgG (Southern Biotechnol-ogy) diluted in 1% milk. Plates were developed with SureBlue TMB mi-crowell peroxidase substrate and TMB stop solution (KPL) and read at450 nm on a MAXline Emax microplate reader (Molecular Devices).Background levels for the assay were determined by incubating serapooled from immunized wild-type mice on uncoated wells.

RESULTS

We screened peritoneal macrophages isolated from third genera-tion (G3) C57BL/6J mice carrying ENU-induced mutations forsusceptibility to ex vivo infection by viruses (MCMV-GFP [27] orthe mouse-adapted influenza virus strain PR8) or a nonreplicatingviral vector (hAd5-F16-GFP [16]). The number of macrophagesinfected by MCMV or hAd5-F16 vector was monitored by flowcytometry to detect GFP-labeled cells; infection by PR8 was deter-mined by the reactivity of macrophages to hemagglutinin (HA)antibody, which also was monitored by flow cytometry. After in-cubation with MCMV-GFP or PR8 for 24 h or with hAd5-F16-GFP for 72 h, the number of infected macrophages from C57BL/6J

Susceptibility to DNA Viruses in a GCN2 Mutant

February 2012 Volume 86 Number 3 jvi.asm.org 1803

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

1804 jvi.asm.org Journal of Virology

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

and G3 mice bearing ENU-induced mutations in homozygousand/or heterozygous form was analyzed.

To validate the effectiveness of the screen, we tested macro-phages from mice with known mutations (Fig. 1A). Macrophagesfrom homozygous Stat1domino (dom) (8) mice were highly permis-sive to infection by all three viruses, confirming the known re-quirement for type I IFN signaling in viral control (17, 28). Incontrast, normal percentages of macrophages from Tlr3�/�,Tlr7�/�, Tlr9CpG1/CpG1 (34), Unc93b13d/3d (35), or Myd88poc/poc

(20) mice were infected by MCMV-GFP, hAd5-F16-GFP, or PR8,indicating that redundant mechanisms for sensing these virusesexist in macrophages, or that requirements for sensing these vi-ruses differ between macrophages and other cell types. IFN-� sig-naling also was dispensable for the control of MCMV-GFP, hAd5-F16-GFP, or PR8 by macrophages, likely because macrophages arenot a significant source of IFN-�.

Macrophages from a total of 4,500 G3 mice were infected withMCMV-GFP, hAd5-F16-GFP, and PR8 (Fig. 1B). Two strains,designated macro-1 and macro-2, produced macrophages thatwere infected by all three viruses with increased frequency relativeto that of C57BL/6J macrophages (Fig. 1B). Both the macro-1 andmacro-2 phenotypes were transmitted recessively. Macrophagesfrom a third strain, atc, were infected with increased frequency byhAd5-F16-GFP and MCMV-GFP (Fig. 1B). The permissiveness ofatc macrophages to hAd5-F16-GFP was similar to that of macro-phages from Stat1dom/dom mice, whereas permissiveness toMCMV-GFP was intermediate between wild-type and Stat1dom/dom

levels. Infection by PR8 was equally well controlled by atc andC57BL/6J macrophages. The atc phenotype also was recessive. As an-other measure of viral control, the amount of type I IFN produced bythe infected macrophages was measured in the culture supernatant.In response to dsDNA, type I IFN production by macro-2 macro-phages was reduced compared to that produced by wild-type cells(Fig. 1C). In contrast, hAd5-F16-GFP induced normal type I IFNproduction by atc macrophages (Fig. 1C).

Because of the importance of type I IFN signaling to the innateantiviral response, the genes encoding STAT1 (Stat1), Isgf3g(Irf9), JAK1 (Jak1), Tyk2 (Tyk2), and the two chains of the type IIFN receptor (Ifnar1 and Ifnar2) were sequenced in macro-1 andmacro-2 mice. An adenine-to-cytosine transversion in exon 8 ofIfnar1, causing the missense mutation T341P in IFNAR1, wasidentified in macro-1 mice (Fig. 2A). An adenine-to-guanine tran-sition in exon 2 of Ifnar2, causing the replacement of the startmethionine with valine, was found in macro-2 mice (Fig. 2B). Thesusceptibility of macro-1 and macro-2 macrophages to infectionby MCMV, human adenovirus, and influenza is consistent with adeficiency of IFNAR signaling, as is the reduced dsDNA- andadenovirus-dependent type I IFN production, which is known tobe regulated by a positive feedback loop in which IFN-� andIFN-�4 induce the expression of the transcription factors IRF7

and IRF8 (25, 36). The detection of IFNAR1 and IFNAR2 defi-ciencies validated the effectiveness of the screen.

The atc phenotype was distinct from those of mice with Ifnar1or Ifnar2 deficiency, and therefore the mutation was mapped bybulk segregation analysis (BSA). Animals with the atc phenotype(C57BL/6J-atc) were outcrossed to C57BL/10J mice, and affectedF1 mice were backcrossed to atc homozygotes (42). Using 40 F2mice with mutant phenotypes and 9 with normal phenotypes,BSA indicated the strongest linkage of the atc mutation with amarker at position 103735349 on chromosome 2 (synthetic loga-rithm of odds [LOD], 6.0) (Fig. 3A). Among nucleotides coveredat least once within the 38 Mb surrounding the marker with peaklinkage (19 Mb upstream and downstream), 327 mutations wereidentified by SOLiD whole-genome sequencing, of which 281were successfully reexamined by conventional capillary sequenc-ing. A single mutation was validated at position 118226389, 14.5

FIG 1 Screen for increased susceptibility to infection by MCMV, human adenovirus, and influenza virus. Thioglycolate-elicited peritoneal macrophages wereinfected with MCMV-GFP (24 h at an MOI of 1), hAd5-F16-GFP (72 h at 104 particles/cell), or PR8 (24 h at 1.5 hemagglutination units/well). (A) Macrophageswere obtained from mice with mutations affecting IFN signaling (left panels) or TLR signaling (right panels). The percentages of macrophages infected aregraphed. Error bars represent standard errors of the means. (B) Macrophages were obtained from homozygous macro-1, macro-2, and atchoum mice. Thepercentages of macrophages infected are graphed. Macrophages from Stat1dom/dom mice were used as susceptible controls. Each data point represents anindividual mouse. Black data points represent G3 mice carrying ENU-induced mutations. (C) Relative concentration of type I IFN, as determined by interferon-stimulated response element (ISRE) reporter assay, in the culture supernatants of homozygous macro-2 or atchoum macrophages treated with dsDNA or infectedwith hAd5-F16-GFP, respectively.

FIG 2 Ifnar1 and Ifnar2 mutations in macro-1 and macro-2 mice. DNA se-quence chromatograms showing the mutated nucleotides in Ifnar1 (adenine tocytosine at position 1115 of cDNA) (A) and Ifnar2 (adenine to guanine atposition 221 of cDNA) (B). The lower panels show the effect of the mutationson the encoded proteins. TM, transmembrane domain; SD, fibronectin typeIII subdomain.

Susceptibility to DNA Viruses in a GCN2 Mutant

February 2012 Volume 86 Number 3 jvi.asm.org 1805

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

Mb from the marker with peak linkage (Fig. 3B). The mutationcorresponds to a thymine-to-cytosine transition of the sixth nu-cleotide of intron 2 of Eif2ak4, encoding GCN2 (Fig. 3C). ThecDNA sequencing of the four largest Eif2ak4 transcripts demon-strated that the mutation invariably results in the skipping of exon2, in some cases along with the skipping of other exons (Fig. 3D).The most abundant transcript lacked exons 2, 3, and 4. No GCN2

expression was detected in Eif2ak4atc/atc macrophages by immu-noblotting (Fig. 3E).

We evaluated the function of the GCN2atc protein upon thestimulation of Eif2ak4atc/atc mouse embryonic fibroblasts with UVradiation, a known activator of GCN2 (9, 18). As expected, UVBresulted in the increased phosphorylation of eIF2� in C57BL/6Jfibroblasts but not in Eif2ak4atc/atc fibroblasts (Fig. 3F), indicatingthat the atc mutation causes the complete loss of GCN2 function,at least where eIF2� phosphorylation is concerned.

To determine whether eIF2� phosphorylation by GCN2 is partof the antiviral response to DNA viruses, we compared eIF2�phosphorylation (eIF2�-P) levels by immunoblotting inC57BL/6J and Eif2ak4atc/atc fibroblasts 0, 8, 16, 24, 32, and 40 hafter infection with MCMV. C57BL/6J fibroblasts displayed min-imal eIF2�-P at each time point up to 32 h but increased eIF2�-P40 h after MCMV infection. In contrast, no eIF2�-P was detect-able in Eif2ak4atc/atc fibroblasts at any time point (Fig. 4A). In aseparate experiment, we measured viral titers at the same timeintervals in the culture supernatant of wild-type and Eif2ak4atc/atc

fibroblasts. Supernatant from Eif2ak4atc/atc fibroblasts displayedtiters similar to those of wild-type cell supernatant at all timepoints examined (Fig. 4B). These findings suggest that eIF2�phosphorylation by GCN2 is not required to prevent virus repli-

FIG 3 Mutation in Eif2ak4 in atc mice. (A) BSA mapping of the atc mutationusing 40 mice with mutant phenotypes and 9 mice with normal phenotypes.LOD scores for 124 markers across the genome were calculated based on BSA.(B) DNA sequence chromatogram showing the mutated nucleotide in Eif2ak4(thymine to cytosine at position 12038 of the genomic DNA sequence). (C)The atc mutation in the context of the encoded protein. RWD, conserveddomain found in RING finger-containing, WD repeat-containing, and yeastDEAD-like helicase proteins; �Kinase, kinase-like domain; HisRS-like,histidyl-tRNA synthetase-like domain; RB/DD, ribosome binding and ho-modimerization domain. (D) RT-PCR products generated from C57BL/6J orEif2ak4atc/atc macrophage RNA using primers spanning the Eif2ak4 cDNA.RT-PCR products were sequenced, and the deduced exon structure of each isindicated. (E) Immunoblot analysis of GCN2 in whole-cell lysates of macro-phages from C57BL/6J or Eif2ak4atc/atc mice. (F) Immunoblot analysis of eIF2�phosphorylation on serine 51 in fibroblasts from C57BL/6J or Eif2ak4atc/atc

mice in response to UVB irradiation.

FIG 4 Increased susceptibility to MCMV infection in Eif2ak4atc/atc mice. (A)Immunoblot analysis of eIF2� phosphorylation (eIF2a-P) on serine 51 inwhole-cell lysates of fibroblasts from C57BL/6J or Eif2ak4atc/atc mice at theindicated times after infection with MCMV (MOI of 3). (B) Viral titer in theculture supernatant of C57BL/6J or Eif2ak4atc/atc fibroblasts at the indicatedtimes after infection with MCMV (MOI of 3). (C) Survival curve of C57BL/6J(n � 39) and Eif2ak4atc/atc mice (n � 68) infected intraperitoneally with 2 �105 PFU of MCMV (P � 0.0041 by log-rank test). Data are pooled from threeindependent experiments with n � 10 and 8, 6 and 12, and 23 and 48 C57BL/6Jand Eif2ak4atc/atc mice, respectively. (D) Antigen-specific IgM or IgG in theserum of C57BL/6J or Eif2ak4atc/atc mice immunized with NP-Ficoll (left) or�-galactosidase (right) analyzed on day 5 or 14, respectively, after immuniza-tion. Each data point represents an individual mouse.

Won et al.

1806 jvi.asm.org Journal of Virology

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

cation and shedding for at least 32 h after infection; an effect onviral titer may lag behind the observation of defective eIF2� phos-phorylation in Eif2ak4atc/atc cells, which first occurs 40 h postin-fection.

We then tested whether the deficiency in eIF2�-P affects sus-ceptibility to MCMV in vivo. C57BL/6J and Eif2ak4atc/atc micewere injected intraperitoneally with 2 � 105 PFU/mouse ofMCMV and observed for sickness. During the 2-week period fol-lowing infection, 11 of 68 Eif2ak4atc/atc mice died, whereas all 39 ofthe C57BL/6J controls remained healthy (P � 0.0041) (Fig. 4C).Thus, GCN2 deficiency increases susceptibility to MCMV infec-tion in mice as it does in cultured macrophages.

The deaths of Eif2ak4atc/atc mice in response to MCMV infec-tion all occurred on or before 9 days postinfection, which is con-sistent with the hypothesis that susceptibility stems from a defectin the innate immune response. However, the expression ofEif2ak4 has been identified as part of a transcriptional signaturehighly correlated with the strength of the adaptive immune re-sponse, in particular the CD8� T-cell response, to the yellow fevervaccine YF-17D (30). We therefore tested the antibody responsesof Eif2ak4atc/atc mice but found that they mounted normal IgGresponses to the T-dependent antigen �-galactosidase and normalIgM responses to the T-independent antigen NP-Ficoll (Fig. 4D).This finding, together with the fact that Rag1-deficient mice sur-vive MCMV infection without incident for several weeks afterinoculation, supports the interpretation that MCMV susceptibil-ity in Eif2ak4atc/atc mice is caused by an innate immune defect.

DISCUSSION

Although the downregulation of protein synthesis as an antiviralresponse generally is attributed to PKR activation, eIF2� phos-phorylation occurs in response to certain viruses even in the ab-sence of PKR function (1, 12, 43). The responses of GCN2- orPERK-deficient mice to viral infections have been reported in onlya few publications (3, 4, 7). By screening macrophages ex vivo, weidentified an Eif2ak4 mutation that caused increased susceptibilityto MCMV and human adenovirus infections. The Eif2ak4atc mu-tation is a single-nucleotide substitution at the sixth position ofEif2ak4 intron 2, which abrogated the splicing of exon 2 to exon 3and resulted in the production of transcripts with aberrant splic-ing from exon 1 to exon 3, exon 4, exon 5, or exon 6. Splicing fromexon 1 to exon 4, exon 5, or exon 6 maintains the correct transla-tional reading frame. We detected no GCN2 protein in cells fromEif2ak4atc/atc mice, suggesting that the translated products are un-stable and degraded.

Experiments focused on MCMV showed that eIF2� was far lessefficiently phosphorylated in Eif2ak4atc/atc fibroblasts than wild-type fibroblasts in response to infection. A previous study (5) re-ported no difference in eIF2� phosphorylation between wild-typeand Eif2ak3�/� Eif2ak4�/� (PERK�/� GCN2�/�) fibroblasts 16 hafter infection with MCMV. At this time, eIF2�-P levels wereequivalent to baseline levels of phosphorylation observed in un-infected cells. These findings are consistent with our data showingthe absence to minimal presence of eIF2�-P in wild-type andEif2ak4atc/atc fibroblasts for at least 32 h after infection, which issimilar to levels in uninfected cells. By 40 h after MCMV infection,however, eIF2�-P was detected in wild-type cells but not inEif2ak4atc/atc cells. Thus, GCN2-dependent eIF2� phosphoryla-tion during the antiviral response to MCMV occurs at least 32 hpostinfection. The relatively late action of GCN2 during the innate

antiviral response may explain the modest susceptibility ofatchoum homozygous mice to MCMV in vivo.

The genomes of both MCMV and human adenovirus encodefactors that function to counter the blockade of translation im-posed by PKR-dependent eIF2� phosphorylation. MCMV pro-teins m142 and m143 bind to PKR, possibly in conjunction withdsRNA, to prevent PKR activation (5). The noncoding virus-associated RNA molecule I (VAI RNA) of adenovirus binds toPKR and blocks its activation (26), while E1B-55K and E4orf6proteins prevent PKR activation through a ubiquitin ligase-dependent mechanism (33). Our findings clearly demonstratethat GCN2 deficiency increases susceptibility to MCMV infectionin vitro and in vivo, albeit only slightly, and raise the possibility thatviral mechanisms also have evolved to oppose eIF2� phosphory-lation specifically by GCN2.

The mechanism by which GCN2 is activated upon MCMVinfection remains unknown. The restricted availability of aminoacids, UV irradiation, and proteasome inhibition are known acti-vators of GCN2, and of these, we hypothesize that viral infection ismost likely to restrict amino acid availability, in that the pool offree amino acids within the cell may be depleted as a result of rapidviral protein synthesis during MCMV and adenovirus infection.This may stimulate GCN2 to downregulate further translation,affecting both cellular and viral proteins. The size of the viral ge-nome does not appear to influence GCN2 activation, as the ge-nomes of MCMV and human adenovirus differ in size by nearly200 kb. The kinetics and the specific mechanisms by which a virustakes control of cellular machinery may be determining factors forGCN2 activation. Understanding the mechanism of GCN2 acti-vation by MCMV infection may also provide insight into the DNAvirus-specific host defense requirement for GCN2. Defenseagainst Sindbis virus, an RNA virus, has been shown to depend onGCN2, which is activated by two noncontiguous regions of theviral genome resembling uncharged tRNA, the natural ligand forGCN2 (4). Such structures apparently are absent from the influ-enza genome, which also fails to drive levels of protein synthesissufficient to activate GCN2 by amino acid depletion.

ACKNOWLEDGMENTS

We are grateful to Shilpi Verma and Chris Benedict for conducting theplaque assays.

This work was supported by National Institutes of Health grant2P01AI070167-06.

REFERENCES1. Abraham N, et al. 1999. Characterization of transgenic mice with targeted

disruption of the catalytic domain of the double-stranded RNA-dependent protein kinase, PKR. J. Biol. Chem. 274:5953–5962.

2. Arnold CN, et al. 2011. Rapid identification of a disease allele in mousethrough whole genome sequencing and bulk segregation analysis. Genet-ics 187:633– 641.

3. Baltzis D, et al. 2004. Resistance to vesicular stomatitis virus infectionrequires a functional cross talk between the eukaryotic translation initia-tion factor 2alpha kinases PERK and PKR. J. Virol. 78:12747–12761.

4. Berlanga JJ, et al. 2006. Antiviral effect of the mammalian translationinitiation factor 2alpha kinase GCN2 against RNA viruses. EMBO J. 25:1730 –1740.

5. Budt M, Niederstadt L, Valchanova RS, Jonjic S, Brune W. 2009.Specific inhibition of the PKR-mediated antiviral response by the murinecytomegalovirus proteins m142 and m143. J. Virol. 83:1260 –1270.

6. Chang HW, Watson JC, Jacobs BL. 1992. The E3L gene of vaccinia virusencodes an inhibitor of the interferon-induced, double-stranded RNA-dependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 89:4825– 4829.

Susceptibility to DNA Viruses in a GCN2 Mutant

February 2012 Volume 86 Number 3 jvi.asm.org 1807

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from

7. Cheng G, Feng Z, He B. 2005. Herpes simplex virus 1 infection activatesthe endoplasmic reticulum resident kinase PERK and mediates eIF-2alphadephosphorylation by the gamma(1)34.5 protein. J. Virol. 79:1379 –1388.

8. Crozat K, et al. 2006. Analysis of the MCMV resistome by ENU mutagen-esis. Mamm. Genome 17:398 – 406.

9. Deng J, et al. 2002. Activation of GCN2 in UV-irradiated cells inhibitstranslation. Curr. Biol. 12:1279 –1286.

10. García MA, Meurs EF, Esteban M. 2007. The dsRNA protein kinase PKR:virus and cell control. Biochimie 89:799 – 811.

11. Georgel P, Du X, Hoebe K, Beutler B. 2008. ENU mutagenesis in mice.Methods Mol. Biol. 415:1–16.

12. Gorchakov R, Frolova E, Williams BR, Rice CM, Frolov I. 2004.PKR-dependent and -independent mechanisms are involved in transla-tional shutoff during Sindbis virus infection. J. Virol. 78:8455– 8467.

13. Han AP, et al. 2001. Heme-regulated eIF2alpha kinase (HRI) is requiredfor translational regulation and survival of erythroid precursors in irondeficiency. EMBO J. 20:6909 – 6918.

14. Harding HP, Zhang Y, Ron D. 1999. Protein translation and folding arecoupled by an endoplasmic-reticulum-resident kinase. Nature 397:271–274.

15. He Y, et al. 2001. Regulation of mRNA translation and cellular signalingby hepatitis C virus nonstructural protein NS5A. J. Virol. 75:5090 –5098.

16. Hsu C, et al. 2005. In vitro dendritic cell infection by pseudotyped ad-enoviral vectors does not correlate with their in vivo immunogenicity.Virology 332:1–7.

17. Hwang SY, et al. 1995. A null mutation in the gene encoding a type Iinterferon receptor component eliminates antiproliferative and antiviralresponses to interferons alpha and beta and alters macrophage responses.Proc. Natl. Acad. Sci. U. S. A. 92:11284 –11288.

18. Jiang HY, Wek RC. 2005. GCN2 phosphorylation of eIF2alpha activatesNF-kappaB in response to UV irradiation. Biochem. J. 385:371–380.

19. Jiang HY, Wek RC. 2005. Phosphorylation of the alpha-subunit of theeukaryotic initiation factor-2 (eIF2alpha) reduces protein synthesis andenhances apoptosis in response to proteasome inhibition. J. Biol. Chem.280:14189 –14202.

20. Jiang Z, et al. 2006. Details of Toll-like receptor:adapter interaction re-vealed by germ-line mutagenesis. Proc. Natl. Acad. Sci. U. S. A. 103:10961–10966.

21. Krishnamoorthy T, Pavitt GD, Zhang F, Dever TE, Hinnebusch AG.2001. Tight binding of the phosphorylated alpha subunit of initiationfactor 2 (eIF2alpha) to the regulatory subunits of guanine nucleotide ex-change factor eIF2B is required for inhibition of translation initiation.Mol. Cell. Biol. 21:5018 –5030.

22. Loewendorf A, et al. 2004. Identification of a mouse cytomegalovirusgene selectively targeting CD86 expression on antigen-presenting cells. J.Virol. 78:13062–13071.

23. Lu L, Han AP, Chen JJ. 2001. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells un-der cytoplasmic stresses. Mol. Cell. Biol. 21:7971–7980.

24. Lu Y, Wambach M, Katze MG, Krug RM. 1995. Binding of the influenzavirus NS1 protein to double-stranded RNA inhibits the activation of theprotein kinase that phosphorylates the elF-2 translation initiation factor.Virology 214:222–228.

25. Marié I, Durbin JE, Levy DE. 1998. Differential viral induction of distinct

interferon-alpha genes by positive feedback through interferon regulatoryfactor-7. EMBO J. 17:6660 – 6669.

26. Mathews MB, Shenk T. 1991. Adenovirus virus-associated RNA andtranslation control. J. Virol. 65:5657–5662.

27. Mathys S, et al. 2003. Dendritic cells under influence of mouse cytomeg-alovirus have a physiologic dual role: to initiate and to restrict T cell acti-vation. J. Infect. Dis. 187:988 –999.

28. Müller U, et al. 1994. Functional role of type I and type II interferons inantiviral defense. Science 264:1918 –1921.

29. Poppers J, Mulvey M, Khoo D, Mohr I. 2000. Inhibition of PKR activa-tion by the proline-rich RNA binding domain of the herpes simplex virustype 1 Us11 protein. J. Virol. 74:11215–11221.

30. Querec TD, et al. 2009. Systems biology approach predicts immunoge-nicity of the yellow fever vaccine in humans. Nat. Immunol. 10:116 –125.

31. Reddehase MJ, et al. 1985. Interstitial murine cytomegalovirus pneumo-nia after irradiation: characterization of cells that limit viral replicationduring established infection of the lungs. J. Virol. 55:264 –273.

32. Rowlands AG, Panniers R, Henshaw EC. 1988. The catalytic mechanismof guanine nucleotide exchange factor action and competitive inhibitionby phosphorylated eukaryotic initiation factor 2. J. Biol. Chem. 263:5526 –5533.

33. Spurgeon ME, Ornelles DA. 2009. The adenovirus E1B 55-kilodalton andE4 open reading frame 6 proteins limit phosphorylation of eIF2alpha dur-ing the late phase of infection. J. Virol. 83:9970 –9982.

34. Tabeta K, et al. 2004. Toll-like receptors 9 and 3 as essential componentsof innate immune defense against mouse cytomegalovirus infection. Proc.Natl. Acad. Sci. U. S. A. 101:3516 –3521.

35. Tabeta K, et al. 2006. The Unc93b1 mutation 3d disrupts exogenousantigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat.Immunol. 7:156 –164.

36. Tailor P, et al. 2007. The feedback phase of type I interferon induction indendritic cells requires interferon regulatory factor 8. Immunity 27:228 –239.

37. Valchanova RS, Picard-Maureau M, Budt M, Brune W. 2006. Murinecytomegalovirus m142 and m143 are both required to block protein ki-nase R-mediated shutdown of protein synthesis. J. Virol. 80:10181–10190.

38. Von Seggern DJ, Huang S, Fleck SK, Stevenson SC, Nemerow GR. 2000.Adenovirus vector pseudotyping in fiber-expressing cell lines: improvedtransduction of Epstein-Barr virus-transformed B cells. J. Virol. 74:354 –362.

39. Wek RC, Cavener DR. 2007. Translational control and the unfoldedprotein response. Antioxid. Redox Signal. 9:2357–2371.

40. Wek RC, Jiang HY, Anthony TG. 2006. Coping with stress: eIF2 kinasesand translational control. Biochem. Soc. Trans. 34:7–11.

41. Wek SA, Zhu S, Wek RC. 1995. The histidyl-tRNA synthetase-relatedsequence in the eIF-2 alpha protein kinase GCN2 interacts with tRNA andis required for activation in response to starvation for different aminoacids. Mol. Cell. Biol. 15:4497– 4506.

42. Xia Y, et al. 2010. Bulk segregation mapping of mutations in closelyrelated strains of mice. Genetics 186:1139 –1146.

43. Yang YL, et al. 1995. Deficient signaling in mice devoid of double-stranded RNA-dependent protein kinase. EMBO J. 14:6095– 6106.

44. Zhang P, et al. 2002. The GCN2 eIF2alpha kinase is required for adapta-tion to amino acid deprivation in mice. Mol. Cell. Biol. 22:6681– 6688.

Won et al.

1808 jvi.asm.org Journal of Virology

on February 13, 2018 by guest

http://jvi.asm.org/

Dow

nloaded from