Species Specificity of Protein Kinase R Antagonism byCytomegalovirus TRS1 Genes

Stephanie J. Child,a Greg Brennan,a Jacquelyn E. Braggin,a,b and Adam P. Geballea,b,c

Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA,a and Departments of Microbiologyb andMedicine,c University of Washington, Seattle, Washington, USA

The host antiviral protein kinase R (PKR) has rapidly evolved during primate evolution, likely in response to challenges posed bymany different viral antagonists, such as the TRS1 gene of cytomegaloviruses (CMVs). In turn, viral antagonists have adapted tochanges in PKR. As a result of this “arms race,” modern TRS1 alleles in CMVs may function differently in cells derived from al-ternative species. We have previously shown that human CMV TRS1 (HuTRS1) blocks the PKR pathway and rescues replicationof a vaccinia virus mutant lacking its major PKR antagonist in human cells. We now demonstrate that HuTRS1 does not havethese activities in Old World monkey cells. Conversely, the rhesus cytomegalovirus homologue of HuTRS1 (RhTRS1) fulfillsthese functions in African green monkey cells, but not rhesus or human cells. Both TRS1 proteins bind to double-stranded RNAand, in the cell types in which they can rescue VV�E3L replication, they also bind to PKR and prevent phosphorylation of the�-subunit of eukaryotic initiation factor 2. However, while HuTRS1 binds to inactive human PKR and prevents its autophos-phorylation, RhTRS1 binds to phosphorylated African green monkey PKR. These studies reveal that evolutionary adaptations inthis critical host defense protein have altered its binding interface in a way that has resulted in a qualitatively altered mechanismof PKR antagonism by viral TRS1 alleles from different CMVs. These results suggest that PKR antagonism is likely one of thefactors that contributes to species specificity of cytomegalovirus replication.

Cytomegaloviruses (CMVs) are generally considered speciesspecific in their replication patterns (33). Human CMV

(HCMV) replicates well in human cells but not in mouse cells,while murine CMV (MCMV) has the opposite host range. How-ever, between more closely related species, the barriers to replica-tion are incomplete. For example, rhesus CMV (RhCMV) canreplicate in human cells as well as rhesus cells (2, 29). Although insome cases modification of a single gene can allow a virus to crossa species barrier (24, 38, 40), the limited host range of CMV rep-lication likely involves multiple viral genes that have adapted tosupport replication in the specific host over millions of years ofcoevolution. Understanding the changes that have occurred inboth host and viral factors has importance for identifying con-served features of the viral life cycle, for assessing the power andlimitations of animal models, and for evaluating the risks andbarriers to cross-species transmission of viruses.

Like other viruses, CMVs have needed to adapt to multiplehost antiviral defenses, including the inhibition of translation bythe protein kinase R (PKR) pathway. PKR is activated by bindingto double-stranded RNA, dimerization, and autophosphorylation(12, 37). Activated PKR then phosphorylates the �-subunit ofeukaryotic initiation factor 2 (eIF2�), resulting in a block to trans-lational initiation and thus to viral replication. Viruses haveevolved multiple different mechanisms for interfering with thishost defense pathway, underscoring the importance of PKR as abarrier to viral replication (34). HCMV encodes two double-stranded RNA binding proteins, TRS1 (HuTRS1) and IRS1, eitherof which is sufficient to prevent activation of the PKR pathway,and at least one of these genes is necessary for HCMV replicationin human fibroblasts (9, 19, 20, 31).

Analyses of the rates of nonsynonymous-to-synonymous sub-stitutions (the dN:dS ratio) in the PKR alleles among primateshave revealed that PKR has been evolving under strong positiveselection, likely as a result of an evolutionary “arms race” with

viral antagonists (14, 36). At one branch point in the primatelineage leading toward rhesus macaques and African green mon-keys (AGMs), PKR acquired a remarkable 22 nonsynonymouschanges but 0 synonymous ones (14). These observations stimu-lated us to investigate the impact that changes in PKR may havehad on the function of antagonists encoded by primate CMVs.

Consistent with the hypothesis that the ability of CMV to an-tagonize PKR may contribute to the host range of viral replication,we found that HuTRS1 blocks PKR activation in human cells butnot in Old World monkey cells. The RhCMV homologue ofHuTRS1 (RhTRS1) is able to block the PKR pathway in some OldWorld monkey cells but not in human cells. RhTRS1 and HuTRS1both bind to double-stranded RNA (dsRNA) and, in the cell typein which each is functional, they bind to PKR. However, HuTRS1binds to inactive human PKR and prevents its phosphorylation,while RhTRS1 binds to and inhibits the eIF2� kinase activity ofAGM PKR after it has been phosphorylated. These results suggestthat evolutionary changes in both PKR and the CMV TRS1 genesresulted in qualitatively different binding interactions and mech-anisms of antagonism.

MATERIALS AND METHODSCells, virus, and infections. Human fibroblasts (HF), telomerase-immortalized HF (HF-tert; obtained from Denise Galloway, FredHutchinson Cancer Research Center [FHCRC]), primary rhesus fibro-blasts (RF; obtained from Klaus Früh and Michael Axthelm, Oregon

Received 29 August 2011 Accepted 13 January 2012

Published ahead of print 25 January 2012

Address correspondence to Adam P. Geballe, ageballe@fhcrc.org.

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

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Health Sciences University), telomerase-immortalized RF (Telo RF; ob-tained from Peter Barry, University of California, Davis [25]), BSC40, andBHK cells were maintained in Dulbecco’s modified Eagle’s medium sup-plemented with 10% NuSerum (BD Biosciences) as previously described(9). HF with PKR expression knocked down (PKR-kd) and controlknockdown HF (ctrl-kd) were produced by cloning of HF-tert lines aftertransduction with lentiviral vectors encoding an shRNA targeting humanPKR or a nonsilencing control shRNA (catalog numbers RHS4430-98844125 and RHS4346, respectively; Open Biosystems).

Vaccinia virus (VV) Copenhagen strain (VC2) (42) and VV�E3L (4),both obtained from Bertram Jacobs (Arizona State University), and VC2-lacZ, VV�E3L�E3L, VV-HuTRS1, VV-RhTRS1, and VC2-GFP (de-scribed below) were propagated and titers were determined in BHK cells(9). �-Galactosidase (�-Gal) activity in infected cells was measured by afluorometric substrate cleavage assay (10). HCMV (strain AD-169; ATCCVR-538) was propagated and titers were determined on HF, and RhCMV(strain 68-1; ATCC VR-677; obtained from Peter Barry, University ofCalifornia, Davis) was propagated and titers were determined on TeloRFcells.

Cell-free translation and yeast two-hybrid plasmids. The plasmidpEQ1100, which expresses enhanced green fluorescent protein (EGFP)with a C-terminal biotinylation signal and adjacent 6�His tag (designated-BH) was previously described (10). pEQ1180 (formerly called pEQ981[19]) expresses full-length HuTRS1-H (-H denotes a 6�His tag). A plas-mid containing RhTRS1-BH (pEQ1215) was prepared by PCR amplifica-tion of RhCMV DNA (provided by Peter Barry, University of California,Davis) using oligos 723 (5=-CCAAAGATCTACCATGCGTCCTCACCGCTCGCCA-3=) and 724 (5=-GCACGGGACGATGAGAACACCAT-3=).The product was digested with BglII and EcoRV and ligated into theBamHI and EcoRV sites in pEQ1068 (8). A series of RhTRS1 deletionmutants was constructed by using pEQ1215 DNA as the template for PCRamplification with the following oligos and cloning the products intopcDNA3.1/V5-His-TOPO (Invitrogen): for RhTRS1(182– 695)-H(pEQ1311), we used oligos 868 (5=-ACCATGAGTCCCTCTCCACAAGAC-3=) and 724; for RhTRS1(347– 695)-H (pEQ1312), we used oligos 869(5=-ACCATGGAATATCTGAGCGAGTGGGCT-3=) and 724; forRhTRS1(1–347)-H (pEQ1313), we used oligos 723 and 870 (5=-TTCCAGCCACGGATGTGTAGT-3=); for RhTRS1(1–548)-H (pEQ1314), weused oligos 723 and 871 (5=-TCTCCCCGGCCGTGTTAAAAA-3=).

Plasmids for yeast two-hybrid assays consisted of kinase-dead PKRalleles fused to the GAL4 transcriptional activation domain (AD) inpGAD424 (Clontech) and TRS1 alleles fused to the GAL4 DNA bindingdomain (BD) in pGBT9 (Clontech). Kinase-dead human PKR-H was firstcloned by PCR amplification of pK296RGFP (obtained from MichaelMathews, University of Medicine and Dentistry of New Jersey [43]) usingoligos 705 (5=-ACCATGGCTGGTGATCTTTCAGCA-3=) and 708 (5=-ACATGTGTGTCGTTCATTTTTCTC-3=) followed by TOPO cloning togenerate pEQ1198. AD-huPKR-H (pEQ1325) was then prepared by PCRamplification of pEQ1198 by using oligos 806 (5=-CCTTGTCGACTCAATGGTGATGGTGATGATG-3=) and 854 (5=-CCTTGGATCCTTATGGCTGGTGATCTTTCAGCA-3=), digesting with BamHI and SalI, and clon-ing the into the same sites in pGAD424.

Kinase-dead rhesus PKR was inserted into pGAD424 by the followingsteps. First, pEQ1260, encoding rhesus PKR(wt)-H, was constructed byPCR amplifying and TOPO cloning of rhesus PKR from pSB819:rhPKR(obtained from Nels Elde, FHCRC) with oligos 772 (5=-ACCATGGCTGGTCATCTTGTACCA-3=) and 773 (5=-ATATGTATGTCGTTTTTTCTCTGGGCT-3=). The HindIII and NotI fragment containing rhesusPKR(wt)-B from pEQ1260 was moved into the same sites in pEQ1068 (8)to generate rhesus PKR(wt)-BH(pEQ1271). A kinase-dead rhesus PKR[RhPKR(K295R)-BH(pEQ1282)] was then constructed using pEQ1271as a template with oligos 800 (5=-GGAAAGACTTACGTTATTAGACGTGTTAAATATAATAGCAAGAAGG-3=) and 801 (5=-CCTTCTTGCTATTATATTTAACACGTCTAATAACGTAAGTCTTTCC-3=) with the Strat-agene QuikChange mutagenesis kit. Finally, AD-rhPKR-H(pEQ1294)

was constructed by PCR amplifying rhesus PKR(K295R)-H frompEQ1282 with oligos 805 (5=-CCTTGGATCCTTATGGCTGGTCATCTTGTACCA-3=) and 806, digesting with BamHI and SalI, and ligating theproduct into the same sites in pGAD424.

To construct kinase-dead AGM PKR(K295R)-H(pEQ1307), the 5=and 3= portions of AGM PKR in pSB819:agmPKR (obtained from NelsElde, FHCRC) were amplified with oligos 801 and 851 (5=-ACCATGGCTGGTGATCTTGCACCA-3=) and oligos 800 and 852 (5=-ACATGTATGTCGTTCCTTTTTCTC-3=), respectively, to introduce the K295R muta-tion. The products of these reactions were mixed and amplified witholigos 851 and 852, and the product was TOPO cloned. AGMPKR(K295R)-H was then PCR amplified from pEQ1307 using oligos 806and 853 (5=-CCTTGGATCCTTATGGCTGGTGATCTTGCACCA-3=),digested with BamHI and SalI, and cloned into the same sites inpGAD424, yielding AD-agmPKR-H(pEQ1328).

Because we found that HuTRS1 was not expressed in Saccharomycescerevisiae, we synthesized yeast codon-optimized forms of both HCMVand RhCMV TRS1 (in pUC57:huTRS1y-H and pUC57:RhTRS1y-H, re-spectively) (GenScript Inc.). To construct BD-huTRS1y-H(pEQ1284),the huTRS1 open reading frame (ORF) was first moved from pUC57:huTRS1y-H into pGBT9 as an EcoRI-PstI fragment. This plasmid wasthen digested with EcoRI, blunted with Klenow, and religated to place thehuTRS1 ORF in the correct reading frame. For BD-RhTRS1y-H(pEQ1297), yeast codon-corrected RhTRS1 was PCR amplified frompUC57:RhTRS1y-H using oligos 824 (5=-GATCGAATTCATGAGACCACATAGATCCCCT-3=) and 825 (5=-GATCGTCGACTTAATGATGATGATGATGATGATG-3=), cut with EcoRI and SalI, and cloned into thesame sites in pGTB9.

VV recombination plasmids and recombinant viruses. The back-bone from which our recombination vectors were derived was pSC11,which contains a lacZ reporter cassette and flanking sequences for recom-bination into the VV thymidine kinase (TK) locus (5). To enable mea-surement of viral replication based on �-Gal activity, we constructed aVC2 virus that expresses �-Gal (VC2�lacZ) by homologous recombina-tion with pSC11. VV�E3L encodes a late promoter:lacZ cassette in placeof the E3L gene; therefore, this virus and the derivatives described belowall express �-Gal during productive replication (4).

Recombination plasmid pEQ854 (9) was derived from pSC11 by re-placement of the lacZ gene with an EGFP-Puro reporter. EGFP-BH wasisolated from pEQ1100 as a BamHI-BclI fragment and inserted intopEQ1131, which is a VV recombination vector derived from pEQ854, byremoving the �-Gal reporter gene by digestion with XhoI and ClaI, blunt-ing with Klenow, and religating. The resulting plasmid, pEQ1145, wasused to generate VC2-GFP by homologous recombination into VC2. Plas-mid pEQ1119, which contains VV E3L-H, was constructed by PCR am-plifying E3L from pMT-E3L (obtained from Bertram Jacobs, ArizonaState University [6]) using oligos 608 (5=-ACCACCATGGCTAAGATCTATATTGACGAG-3=) and 609 (5=-GAATCTAATGATGACGTAACC-3=), followed by TOPO cloning. To make VV�E3L�E3L (VVeq1127), aBamHI-BclI fragment from pEQ1119 containing E3L-H was ligated intothe BamHI site of pEQ854. The resulting recombination vector(pEQ1127) was then used to construct VV�E3L�E3L (VVeq1127) byhomologous recombination into VV�E3L.

The VV-HuTRS1 (VVeq1148) recombinant was constructed by re-placing the BamHI-BsrG1 fragment containing EGFP in pEQ1145 withTRS1 codons 1 to 760 from pEQ876 (19) followed by recombination intothe TK locus of VV�E3L. To generate VV-RhTRS1, we first madepEQ1135, in which the EFGP-puro cassette was replaced with a neomycinresistance gene, by amplifying pcDNA3.1/V5/His using oligos 624 (5=-GATCCTCGAGACCATGGTTGAACAAGATGGATTGCAC-3=) and 625(5=-CTAGATCGATACCACAACTAGAATGCAGTG-3=), digesting theproduct with XhoI and ClaI, and inserting it into the same sites inpEQ854. We then digested pEQ1135 with BamHI and EcoRI, blunted theends with Klenow, and inserted an RhTRS1-BH cassette derived frompEQ1215 by HindIII digestion, blunting, and PmeI digestion, yielding

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pEQ1233. VV-RhTRS1 was produced by homologous recombination ofpEQ1233 into VV�E3L to generate VV-RhTRS1 (VVeq1233).

Immunoblot analyses. Cells were mock infected or infected withCMV or VV (multiplicity of infection [MOI], 3). At various times postin-fection, the cells were washed with phosphate-buffered saline (PBS) andthen lysed in 2% sodium dodecyl sulfate (SDS). Equivalent amounts of thesamples were separated on 10% SDS-polyacrylamide gels, transferred topolyvinylidene difluoride (PVDF) membranes, and probed with one ofthe following antibodies: Penta-His (Qiagen), PKR (sc-6282 [Santa CruzBiotechnology, Inc.] or 07-151 [Upstate Biotechnology]), phospho-PKR(T446; 1120-1; Epitomics), actin (A2066; Sigma), TRS1 �999 (31), eIF2�or phospho-eIF2� (Ser51) antibody (both from Cell Signaling Technol-ogy, catalog numbers 9722 and 9721, respectively). All purchased anti-bodies were used according to the manufacturer’s recommendations. Forall immunoblot analyses, proteins were detected using the Western Starchemiluminescent detection system (Applied Biosystems) according tothe manufacturer’s recommendations.

Metabolic labeling. HF, RF, and BSC40 cells were mock infected orinfected with the indicated viruses. At 24 h postinfection, the cells werelabeled for 4 h with 100 �Ci/ml [35S]methionine (Easytag express proteinlabeling mix; PerkinElmer) in medium lacking methionine. The cells werethen washed in PBS and lysed in 2% SDS. Equivalent amounts of protein(50 �g) from each sample were separated on 10% SDS-polyacrylamidegels, dried, and visualized by autoradiography.

Double-stranded RNA binding assays. dsRNA [poly(rI · rC)]-agarose beads were prepared as previously described (28). The TnT QuickCoupled transcription/translation system (Promega) containing 1�Ci/ml [35S]methionine was used to produce in vitro-translated proteinsfrom the indicated plasmids. Five microliters of lysate in 250 �l of bufferA (100 mM KCl, 20 mM HEPES [pH 7.5], 10% glycerol, 5 mM MgOAc, 1mM dithiothreitol, 1 mM benzamidine [Sigma] plus 1% NP-40) was in-cubated with dsRNA-agarose beads (plus carrier Sepharose CL-6B[Sigma-Aldrich]) or with Sepharose CL-6B alone as a binding control for1 h at 4°C on a rotating mixer. After binding, the beads were pelleted(16,000 � g; 30 s) and washed in buffer A (0.7 ml) 3 to 4 times. Thesamples were denatured at 95°C for 5 min, separated on 10% SDS-polyacrylamide gels, and analyzed by autoradiography. For analyses ofdsRNA binding proteins in cells, lysates were made by washing the cellsonce in PBS, then lysing them directly on the culture plate in buffer A. Thelysates were collected by scraping and incubated on a rotating mixer for 15min at 4°C, the nuclei were pelleted, and the remaining supernatant wasincubated with dsRNA-agarose beads as described above. For competi-tion assays, extracts were preincubated in buffer A with free poly(rI · rC)competitor (200 �g) for 30 min on a rotating mixer prior to addition ofdsRNA-agarose beads or control Sepharose CL-6B beads (Sigma-Aldrich). The proteins were separated by SDS-PAGE, transferred toPVDF, and probed with TRS1 �999 antiserum. Lysate corresponding to3% of the amount used for each binding reaction was analyzed alongsideeach set of binding reactions.

Yeast two-hybrid assays. A yeast two-hybrid assay (16) was used toassay PKR-TRS1 interactions. Saccharomyces cerevisiae pJ69-4� (obtainedfrom Nels Elde, FHCRC [23]), a yeast strain in which the interaction ofAD and BD fusion proteins results in activation of His3 and �-Gal re-porter genes, was transformed with the indicated plasmids by using high-

efficiency lithium-acetate transformation (17). Transformants wereplated onto YC medium (yeast complete minimal medium with aminoacids containing 2% glucose) -Leu -Trp and incubated for 3 days at 30°C.Colonies were then streaked onto YC -Leu -Trp and YC -Leu -Trp -Hisplates and incubated at 30°C for 3 to 4 days, after which growth of thetransformants was analyzed visually. Protein-protein interactions werealso quantified by measuring �-Gal expression, essentially as described inthe Clontech user manual for �-Gal detection in yeast extracts (PT3037-1). Briefly, 2-ml aliquots from overnight cultures grown in liquid YC -Leu-Trp medium at 30°C were brought up to 5 ml with yeast extract-peptone-dextrose (YEPD) medium and incubated for an additional 6 to 8 h. Ap-proximately 3 ml of each sample was pelleted (16,000 � g; 30 s), washed in1.5 ml of Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1mM MgSO4, 50 mM �-mercaptoethanol), and then resuspended in 300�l of Z-buffer. After freeze-thawing three times, the supernatant was usedfor �-Gal assays and for determining protein concentrations.

DNA sequences and alignment. Predicted amino acid sequences forHuTRS1 and RhTRS1 (GenBank accession numbers FJ616285 andAY186194, respectively) were aligned in Geneious v4.8.5 using the Blo-sum62 cost matrix with a default gap open penalty of 12 and gap extensionpenalty of 3. The alignment was refined by hand to maximize pairwiseidentity.

Nucleotide sequence accession numbers. The sequences of the yeastcodon-optimized HuTRS1 and RhTRS1 genes HuTRS1y-H andRhTRS1y-H, respectively, have been deposited in GenBank and assignedaccession numbers JQ 62772 and JQ 27773.

RESULTSSpecies-specific replication of VV-HuTRS1 and VV-RhTRS1.The observation that RhCMV can replicate in human cells (2, 29)suggests that it encodes an antagonist capable of inhibiting humanPKR. HCMV encodes two such proteins, HuTRS1 and IRS1, but awhole-genome analysis of RhCMV identified a homologue of onlyHuTRS1 (RhTRS1) (21). Overall amino acid identity betweenHuTRS1 and RhTRS1 was 35%; however, this homology was notevenly distributed, and some regions shared much higher homol-ogy, such as the amino-terminal dsRNA binding domain identi-fied in HuTRS1 (�53%) (Fig. 1). Because of the sequence homol-ogy between HuTRS1 and RhTRS1, we hypothesized that RhTRS1is likely a PKR antagonist. PKR antagonists from heterologoussystems can rescue replication of a VV mutant lacking its ownmajor PKR antagonist, E3L (VV�E3L) (4, 9). Therefore, we con-structed VV�E3L recombinants containing HuTRS1 (VV-HuTRS1) or RhTRS1 (VV-RhTRS1) to study the host range andmechanism of action of these TRS1 alleles.

We first infected HF, RF, or AGM cells (BSC40) with VV con-taining wild-type E3L (VC2�lacZ), VV�E3L, VV-HuTRS1, orVV-RhTRS1. Each of these viruses contains a lacZ cassette, allow-ing measurement of �-Gal activity for monitoring viral replica-tion (7). As expected, VC2�lacZ replicated relatively well com-pared to VV�E3L in all three cell types (Fig. 2). VV-HuTRS1

FIG 1 Predicted amino acid sequence alignment of HCMV Towne TRS1 and RhCMV 68-1 TRS1. Black vertical bars indicate amino acid identity, gray verticalbars indicate amino acid differences, and horizontal regions indicate gaps. The previously determined dsRNA binding domain and region required for interactingwith PKR in HuTRS1 are noted by bars over the alignment. Arrows below the alignment indicate the sites of RhTRS1 truncation mutants discussed later.

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replicated well in HF but not in RF or BSC40 cells. In contrast,VV-RhTRS1 replicated well only in BSC40 cells and not in HF or,unexpectedly, not even in RF. Thus, the two TRS1 alleles demon-strated species-specific differences in their abilities to supportVV�E3L replication.

To clarify the basis for these replication patterns, we monitoredoverall protein synthesis at 24 h postinfection (hpi) by metaboliclabeling, SDS-PAGE, and autoradiography. All cells infected withVV�E3L synthesized very low levels of new protein, while thoseinfected with VC2�lacZ produced a pattern indicative of ongoingviral protein synthesis at 24 hpi (Fig. 2B). After infection withVV-HuTRS1, HF continued to synthesize proteins while RF andBSC40 did not. After VV-RhTRS1 infection, BSC40 cells continuedto synthesize viral proteins while HF and RF did not. Thus, theability of each TRS1 allele to rescue VV�E3L replication corre-sponded to its ability to support continued protein synthesis, sug-gesting that the restricted replication of VV-HuTRS1 in RF andBSC40 cells and of VV-RhTRS1 in HF and RF may be due to theirfailure to block the PKR pathway.

Species-specific effects of TRS1 proteins are mediated by thePKR pathway. The replication patterns noted in Fig. 2 could havebeen due to differences in expression of the alternative TRS1 pro-

teins or to differences in their activities in the alternative cell types.To distinguish between these possibilities, we first evaluated ex-pression of the TRS1 proteins in an immunoblot assay, makinguse of the C-terminal His tag present on each protein. In thisexperiment we used a recombinant, VV�E3L�E3L, which con-tains a His-tagged E3L gene inserted into the thymidine kinaselocus, in place of VC2�lacZ, to enable comparison of expressionamong the various PKR antagonists.

As in the previous experiment (Fig. 2), VV-HuTRS1 replicatedwell in HF but not BSC40 cells, while VV-RhTRS1 had the oppositepattern (Fig. 3A). VV�E3L�E3L replicated moderately well inHF. Although in this experiment it replicated to a low level inBSC40 cells, this virus consistently replicated to a greater extentthan the background level of VV�E3L replication (data notshown). Immunoblot assays revealed that HuTRS1 was easily de-tectable in HF but was only faintly detectable in VV-HuTRS1-infected BSC40 cells at 24 hpi (Fig. 3B, bottom panel). Conversely,RhTRS1 was detectable in BSC40 cells but not in HF after infectionwith VV-RhTRS1. E3L was detectable in both cell types infectedwith VV�E3L�E3L. Thus, expression of the PKR antagonists cor-related with rescue of VV replication.

We next explored the possibility that the cause of the limitedexpression of HuTRS1 in BSC40 cells and of RhTRS1 in HF wasthat the TRS1 variants were unable to block PKR in the nonper-missive cell types. When we examined the expression patterns at 6hpi, we detected similar amounts of HuTRS1 and RhTRS1 inBSC40 cells. RhTRS1 was also detectable at 6 hpi in HF at a levelonly slightly lower than HuTRS1. These results revealed that bothVV-HuTRS1 and VV-RhTRS1 enter both cell types and expressthe encoded TRS1 proteins early during infection. However, as aresult of an apparent inability of HuTRS1 to prevent the shutoff ofprotein synthesis in BSC40 cells and of RhTRS1 to perform thisfunction in HF (Fig. 2), the TRS1 proteins fail to accumulate andare absent or nearly absent by 24 hpi in these nonpermissive cells.

We have focused our experiments on HF and BSC40 cells, inwhich HuTRS1 and RhTRS1 have divergent properties, but wealso evaluated expression of these proteins in RF, in which neitherone rescues VV�E3L replication or prevents shutoff of proteinsynthesis (Fig. 2). At both 6 and 24 hpi, VV-HuTRS1 and VV-RhTRS1 expressed barely detectable levels of HuTRS1 andRhTRS1, respectively, compared to the expression of E3L fromVV�E3L�E3L (Fig. 3C). Although there may be a mechanismthat stabilizes specific TRS1 proteins in specific cell types, theseresults are most consistent with the conclusion that TRS1 proteinsthat cannot block the shutoff of protein synthesis fail to accumu-late due to their reduced production during infection.

If the failure to block PKR activation accounts for the lack ofRhTRS1 accumulation in HF, then knocking down PKR in HFshould augment RhTRS1 expression. To test this prediction, wetransduced HF-tert cells with retroviral vectors containing anshRNA targeting PKR or a control shRNA, prepared clonal deriv-atives of these cells, and documented the successful knockdown ofPKR in an immunoblot assay (Fig. 3D). In the PKR knockdowncells, all viruses, including VV�E3L and VV-RhTRS1, replicatedto a similar extent (Fig. 3A). Notably, RhTRS1 was expressed at ahigh level even at 24 hpi in these cells (Fig. 3B). The control knock-down cells behaved like the parental HF. All the viruses replicatedwell and expressed the His-tagged proteins in BHK cells, which areknown to be fully permissive for VV�E3L (27).

These results suggest that RhTRS1 cannot block human PKR,

FIG 2 Replication of VV-HuTRS1 and VV-RhTRS1 is cell type specific. (A)Measurement of viral replication in HF, RF, and BSC40 cells. Triplicate wells ofeach cell type were mock infected or infected (MOI, 3) with VC2�lacZ (VC2),VV-HuTRS1, VV-RhTRS1, or VV�E3L. At 24 hpi, viral replication was quan-tified by measurement of �-Gal as described in Materials and Methods. Themean enzyme activities (� standard deviations) are shown. (B) Analysis oftotal protein synthesis by metabolic labeling of infected HF, RF, and BSC40

cells. The same mock-infected or infected cells analyzed in panel A were pulse-labeled with [35S]methionine as described in Materials and Methods, afterwhich cell lysates were prepared and the proteins analyzed by SDS-PAGE andautoradiography.

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that HuTRS1 cannot block AGM PKR, and that neither TRS1blocks rhesus PKR. As a result, protein synthesis ceases and littleor no TRS1 accumulates by 24 hpi in the nonpermissive cell types.The failure of RhTRS1 to inhibit human or rhesus PKR was unex-pected in light of the ability of RhCMV to replicate in HF and RF.We therefore investigated whether the functions necessary for in-hibition of human PKR by HuTRS1 were conserved in RhTRS1.

RhTRS1 binds to dsRNA. Previous studies have suggested thatHuTRS1 needs to bind to both dsRNA and to PKR in order torescue VV�E3L replication (19, 20). Since RhTRS1 is unable torescue VV�E3L replication in human cells (Fig. 2 and 3), we hy-pothesized that it might lack one of these activities.

To evaluate whether RhTRS1, as expressed in human cellsby RhCMV infection, binds to dsRNA, we prepared lysates ofHF after mock infection or infection with RhCMV or HCMV.We assessed the dsRNA binding ability of the TRS1 proteins byimmunoblot assay of proteins that bound to poly(I · C)-agarose beads in the presence or absence of free competitorpoly(I · C) (Fig. 4A). Like HuTRS1, RhTRS1 was detectable inthe cell lysates when we used a polyclonal serum raised toHuTRS1 (31). Pull-down assays showed that RhTRS1 andHuTRS1 each bound to the dsRNA beads and that the bindingwas competed by free poly(I · C).

These results demonstrate that RhTRS1, as expressed in HF

FIG 3 Cell-type-specific differences in VV-HuTRS1 and VV-RhTRS1 replicationare mediated by PKR. (A) Viral replication in various cell types. The indicated cellswere mock infected or infected with VV�E3L�E3L, VV-HuTRS1, VV-RhTRS1,or VV�E3L (MOI, 3), and viral replication was measured with a �-Gal activityassay as described in Materials and Methods. Mean enzyme activity (� standarddeviation) for triplicate wells is shown. (B) Expression of virally encoded His-tagged proteins in different cell types. Protein lysates were collected from the in-fected cells shown in panel A at 24 hpi (bottom blot) or from another set of wells at6 hpi (upper blot) and were subjected to immunoblot analysis using anti-Hisantibody as described in Materials and Methods. Arrowheads (right) indicateHuTRS1 and RhTRS1. (C) Expression of HuTRS1 and RhTRS1 in RFs. Lysates fromRFcells infectedas indicatedwerepreparedat6and24hpi,andproteinexpressionwasanalyzed by immunoblotting with anti-His antibody. (D) PKR expression from pa-rental HF-tert cells (HF) or from cells stably transduced with either a PKR-targetingshRNA (PKR-kd) or a control shRNA (Ctrl-kd). Cell lysates were prepared and ana-lyzed by immunoblotting with PKR (sc-6282) and actin antibodies.

FIG 4 RhTRS1 binds to dsRNA. (A) dsRNA binding by HuTRS1 and RhTRS1following CMV infection. HFs were infected with HCMV or RhCMV (MOI,3). Cell lysates prepared at 48 hpi were preincubated with no competitor (-) orwith free poly(I·C) competitor (�) and then incubated with dsRNA-agarosebeads. Bound proteins and total cell lysates (Lys) were analyzed by immuno-blot assay using TRS1 antiserum as described in Materials and Methods. (B)Binding of cell-free translated RhTRS1 to dsRNA-agarose. Full-lengthRhTRS1, the indicated deletion mutants, and the nonbinding control, GFP,were in vitro translated in the presence of [35S]methionine, then bound todsRNA-agarose (I:C) or naked control beads (NB), and washed, and thebound proteins were analyzed alongside total cell-free lysate (Lys) by SDS-PAGE and autoradiography as described in Materials and Methods.

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after RhCMV infection, can bind to dsRNA. However, as is thecase for MCMV (10), it is possible that dsRNA binding byRhTRS1 may require a second RhCMV protein that is pro-duced by RhCMV infection but not by VV-RhTRS1 infection.Therefore, we tested the dsRNA binding properties of RhTRS1in the absence of other viral proteins by cell-free translation ofRhTRS1 followed by dsRNA binding assays. In these experi-ments, full-length RhTRS1 bound to poly(I · C)-agarose beadsbut not to control beads, indicating that, like HuTRS1, RhTRS1alone can bind to dsRNA (Fig. 4B).

RhTRS1 and HuTRS1 are most similar in sequence in theiramino-terminal regions, which is where the HuTRS1 dsRNAbinding domain is localized (Fig. 1). To assess whether the func-tional organization of RhTRS1 is similar to HuTRS1, we testedseveral N- and C-terminal deletions for dsRNA binding activity(Fig. 4B). Deletion of the N-terminal region to codon 182 or be-yond eliminated dsRNA binding. Deletion of the C terminus tocodon 548 slightly reduced binding, while deletion to codon 347greatly reduced but did not entirely eliminate binding. Thus, as inHuTRS1, the N-terminal region of RhTRS1 seems to contain theresidues required for dsRNA binding.

These data indicate that, like HuTRS1, RhTRS1 binds todsRNA. Thus, the different abilities of HuTRS1 and RhTRS1 toblock the PKR pathway and to support VV�E3L replication inhuman cells are not easily explained by any apparent difference intheir dsRNA binding activities.

RhTRS1 does not bind to kinase-dead PKR. An alternativeexplanation for the failure of RhTRS1 to rescue VV�E3L replica-tion in HF is that, unlike HuTRS1, it does not bind to human PKR.To test this possibility we cloned yeast codon-optimized RhTRS1and HuTRS1 genes into the yeast two-hybrid binding domainvector pGBT9. Because wild-type PKR genes from many primatespecies inhibit yeast growth (14), we used kinase-dead point mu-tants of the human, rhesus, and AGM PKR genes cloned into theyeast two-hybrid activation domain vector pGAD424. All bindingand activation domain plasmid double transformants grew wellon control (YC -Leu -Trp) plates (data not shown). Consistentwith previous coimmunoprecipitation assays in mammalian cells(20), HuTRS1 directly interacted with human PKR as assessedboth by yeast growth on His-minus plates (Fig. 5A) and by expres-sion of �-Gal (Fig. 5B) from the lacZ reporter gene in the yeaststrain. HuTRS1 also appeared to bind inefficiently to rhesus PKRbut not at all to AGM PKR or to the empty activation domainvector. Despite a small amount of growth of transformants con-taining RhTRS1 and either human or rhesus PKR on His-minusplates, these yeast cells did not express �-Gal above backgroundlevels. Surprisingly, RhTRS1 did not bind to AGM PKR. Thus,RhTRS1 does not bind strongly to kinase-dead PKR from any ofthe tested primates.

Differing mechanisms of PKR pathway inhibition byHuTRS1 and RhTRS1. The puzzling finding that RhTRS1 rescuesprotein synthesis and VV�E3L replication in AGM cells but isunable to bind to kinase-dead AGM PKR in a yeast two-hybridassay suggested that the mechanism by which RhTRS1 functionsin AGM cells differs from that of HuTRS1 in human cells. There-fore, we analyzed the PKR activation pathway in more detail in HFand BSC40 cells after infection with VV-HuTRS1, VV-RhTRS1,and control viruses.

We first examined the activation of PKR in these lysates byimmunoblot assay, using an antibody directed against human

phospho-PKR (T446), which also reacts with the phosphorylatedAGM PKR. HuTRS1 prevented PKR phosphorylation in humancells but not in BSC40 cells (Fig. 6). Consistent with its failure tobind either human or AGM PKR (Fig. 5), RhTRS1 did not preventPKR phosphorylation in HF or BSC40 cells.

We next examined the impact of PKR activation on the nextstep in the pathway, phosphorylation of eIF2�. As predicted basedon the presence of activated PKR, phosphorylated eIF2� accumu-lated in BSC40 cells infected with VV-HuTRS1 and in HF infectedwith VV-RhTRS1, but not in HF infected with VV-HuTRS1 (Fig.6). The abundance of phosphorylated eIF2� in HF after infectionwith VV-RhTRS1 appeared to be somewhat less than afterVV�E3L infection in this experiment, but that difference was not

FIG 5 RhTRS1 does not bind to PKR in the yeast two-hybrid system. (A) Yeasttwo-hybrid analysis of S. cerevisiae strain pJ69-4� transformed with the indi-cated binding domain plasmids (outer labels, HuTRS1, RhTRS1, and the par-ent vector, pGTB9) and activation domain expression vectors (PKR alleles andthe vector control, pGAD424) by growth on -His plates. (B) Quantification ofprotein-protein interactions by �-Gal expression. Yeast double transformantswere grown in -Leu, -Trp medium overnight, then in YEPD medium for anadditional 6 to 8 h. Lysates were prepared and assayed for �-Gal as described inMaterials and Methods.

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observed in other similar experiments, and in all cases the eIF2�phosphate levels were much higher after infection with VV-RhTRS1 compared to mock infection or infection with VC2- orVV-HuTRS1. Notably, despite its failure to block PKR phosphor-ylation, RhTRS1 inhibited eIF2� phosphorylation in BSC40 cells.This effect on eIF2� is consistent with RhTRS1’s ability to rescueVV�E3L replication and protein synthesis in BSC40 cells (Fig. 2and 3). These results reveal that RhTRS1 inhibits AGM PKR in aqualitatively different manner than the way HuTRS1 inhibits hu-man PKR.

The observation that RhTRS1 is unable to block PKR phos-phorylation but does inhibit phosphorylation of eIF2� led us toinvestigate the possibility that RhTRS1, unlike HuTRS1, mightbind only to the activated form of PKR. In line with this hypoth-esis, phosphorylated AGM PKR clearly associated with RhTRS1 inBSC40 cells infected with VV-RhTRS1 (Fig. 7, lane 10, PKR-P pulldown). To evaluate whether RhTRS1 binds to inactive AGM PKR,we coinfected BSC40 cells with both VV-RhTRS1 and VC2-GFP(lane 11). Under these conditions, PKR remained unphosphoryl-ated, presumably due to expression of E3L by VC2-GFP. Althoughexpression of RhTRS1 was also reduced in the coinfected cells(likely due to the lower MOI of VV-RhTRS1 used and competi-tion between the two viruses), the complete absence of PKR bind-ing to RhTRS1 (lane 11, PKR-tot) suggested that RhTRS1 does notbind to inactive AGM PKR in mammalian cells. We cannot ex-clude the possibility that under these conditions E3L binds toAGM PKR and sterically prevents RhTRS1 from also binding.However, together with the yeast two-hybrid results, these datasupport a model in which RhTRS1 binds to AGM PKR only afterit has undergone autophosphorylation and is then able to blockthe eIF2� kinase reaction, consequently allowing continued pro-tein synthesis and viral replication.

We used a similar strategy to examine the PKR binding prop-erties of HuTRS1 in mammalian cells. Consistent with the priorexperiment (Fig. 6), HuTRS1 blocked PKR phosphorylation to alarge extent in HF (Fig. 7, lane 1 versus 6, PKR-P). However, it did

also bind to the small amount of phosphorylated human PKR thataccumulated in VV-huTRS1-infected cells (lane 1). Coinfection ofHF with VV-huTRS1 and VC2-GFP reduced phosphorylation ofhuman PKR to background levels and reduced expressionof HuTRS1, but the HuTRS1 that was expressed bound to humanPKR (lane 2, PKR-tot). These results are consistent with cotrans-fection experiments in which we consistently found that kinase-dead PKR bound to human TRS1 (data not shown). In BSC40 cells,HuTRS1 bound very weakly to phosphorylated AGM PKR (lane 8,PKR-P). When phosphorylation of AGM PKR was blocked bycoinfection with VC2-GFP, we detected no binding to AGM PKR(lane 9, PKR-tot pull down).

Together these results show that HuTRS1 and RhTRS1 havediffering binding properties and activities. HuTRS1 binds to inac-tive human PKR and prevents its autophosphorylation. HuTRS1also binds to phosphorylated human PKR, and to a slight extent toAGM PKR, but at least in the latter case its binding is unable toblock the eIF2� kinase activity of AGM PKR. In contrast, RhTRS1does not bind to or block autophosphorylation of AGM or humanPKR, but it is able to bind to phosphorylated AGM PKR andblocks its eIF2� kinase activity.

DISCUSSION

Pressure from pathogenic viruses can drive the rapid evolution ofhost genes that have antiviral activities (15). In response, viralantagonists of these genes also undergo rapid evolution. For ex-ample, changes in the PKR gene during primate evolution haveresulted in modern alleles that have greatly varying sensitivities toinhibition by the poxvirus eIF2�-mimic K3L, which is itself underpositive selection (14). Multiple codons in PKR have been evolv-ing rapidly, likely in response to pressure from PKR antagonistsencoded by extinct viruses and ancestors of extant viruses.

This evolutionary perspective led us to investigate the impactthat PKR adaptations may have had on the species-specific infec-tivities of CMVs. Since CMVs are believed to have coevolved withtheir host species, each may have contributed to, as well as adaptedto, changes in PKR. Consistent with this view, HCMV replicates inhuman cells but not in Old World monkey cells (data not shown),and HuTRS1 inhibits human but not Old World monkey PKR(Fig. 2 and 6). The finding that HuTRS1 binds to human PKR andblocks its autophosphorylation (20) (Fig. 5 to 7) can account forits ability to block eIF2� phosphorylation and to maintain proteinsynthesis in cells infected with HCMV and VV mutants that lackother PKR antagonists (11, 31) (Fig. 2 and 6). We also found thatVV-HuTRS1 replicates well in cells from other hominoids, in-cluding chimpanzees, orangutans, gorillas, and gibbons (data notshown). In contrast, HuTRS1 does not bind to rhesus or AGMPKR in yeast two-hybrid assays or in AGM cells (Fig. 5 and 7) andis unable to block the PKR pathway in rhesus or AGM cells (Fig. 2,6, and 7). Taken together, these data suggest that the ability ofHuTRS1 to inhibit PKR activation is hominoid lineage specific.One or more of the many changes that arose during the rapidevolution of PKR in Old World monkeys likely accounts for theirresistance to HuTRS1 binding and antagonism and may contrib-ute to the inability of HCMV to replicate in Old World monkeycells.

Unlike HCMV, RhCMV replicates in both Old World monkeycells and human cells (2, 29). This fact, along with studies showingthat the PKR antagonists encoded by both HCMV and MCMV areessential in order for the viruses to replicate at all in cell culture

FIG 6 Effects of HuTRS1 and RhTRS1 on PKR and eIF2� phosphorylation.HF and BSC40 cells were mock infected or infected with VC2, VV-HuTRS1,VV-RhTRS1, or VV�E3L. At 48 hpi the cells were lysed, and equivalentamounts of protein were analyzed by immunoblotting with antibodies di-rected against total and phospho-PKR, total and phospho-eIF2�, and actin.

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(10, 31, 32, 44), suggests that RhCMV likely has the ability to blockhuman PKR. Yet RhTRS1, the predicted RhCMV antagonistbased on homology to HCMV, is unable to block the PKR path-way in human cells. Surprisingly, RhTRS1 also appears unable toblock rhesus PKR, as judged by its inability to rescue protein syn-thesis or VV�E3L replication in RF. However, RhTRS1 is able toblock eIF2� phosphorylation in BSC40 cells (Fig. 6) and can rescueVV�E3L replication in several other AGM cell lines (data notshown). We have not ruled out the possibility that RhTRS1 acts inBSC40 cells by blocking another of the cellular eIF2� kinases.However, VV�E3L replicates in cells in which PKR has beenknocked out or down (45, 46) (Fig. 3), and RhTRS1 does bind toactivated AGM PKR (Fig. 7). Thus, our results support the con-clusion that RhTRS1 functions by blocking PKR in AGM cells, butnot in human cells or even in cells from its host species, the rhesusmacaque. We found that VV-RhTRS1 does not replicate well infive other RF lines (provided by Peter Barry) (data not shown), soit is unlikely that the RF cell line we used in these experiments is in

some way not representative of RF. However, we have not yettested VV-RhTRS1 in a panel of primary AGM cells in order toclarify whether its ability to replicate in BSC40 cells is a property ofthe PKR gene or somehow related to the fact that these are anestablished cell line. HuTRS1 functions well in both primary andtransformed human cells (9, 19, 31), leading us to hypothesize thatthe ability of TRS1 alleles to antagonize PKR tracks more closelywith the PKR gene than with the transformation state of the cells,but additional studies are needed to clarify this issue.

Another possibility is that RhCMV really is an AGM CMVisolate. In this regard, RhCMV strain 68-1 was isolated from rhe-sus macaques that were housed with AGM (2). However, thisstrain clearly replicates in rhesus macaques (30, 41), and its se-quence differs substantially from that reported for AGM CMV (1,3). Thus, it seems most likely that RhCMV really is a rhesus ma-caque virus, albeit one that may be able to cross species barriers.

The fact that RhTRS1 appears unable to block PKR in humanor rhesus cells raises the question as to how RhCMV is able to

FIG 7 RhTRS1 binds to activated AGM PKR. HF and BSC40 cells were mock infected or infected with VV-HuTRS1, VV-RhTRS1, VC2-GFP, or VV�E3L (MOI,3) or coinfected with VV-HuTRS1 or VV-RhTRS1 (MOI, 2) and VC2-GFP (MOI, 4) as indicated. At 24 hpi cell lysates were prepared, and equivalent amountsof each lysate were incubated with nickel-agarose beads. Cell lysates and bound proteins were then analyzed by immunoblotting with antibodies directed againstphospho-PKR, total PKR, His, and actin. Arrowheads indicate bound total PKR.

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replicate in these cells. It is possible that VV�E3L-based assays donot accurately model events occurring during CMV infections.For example, perhaps RhCMV infection produces a relativelysmall amount of dsRNA or for some other reason only activatesPKR to a low level compared to VV-RhTRS1 infection, and thuseven weak antagonism of human or rhesus PKR by RhTRS1 maybe sufficient to support replication of RhCMV. This explanationwould not account for the differences in host range of VV-RhTRS1 and VV-HuTRS1, which likely produce similar amountsof dsRNA. Even if VV requires a more potent PKR antagonist thando CMVs, our experiments illuminate a clear difference in thepotencies of HuTRS1 and RhTRS1 in cells from different species.Another explanation for the ability of RhCMV to replicate in hu-man and rhesus cells while VV-RhTRS1 does not is that RhCMVmight encode a second factor that acts with RhTRS1 to blockhuman and rhesus PKR, analogous to the MCMV system in whichtwo viral genes, m142 and m143, are both required to block thePKR pathway and for viral replication in mouse cells (10, 31, 32,44). It is also possible that RhCMV encodes another gene (orgenes) that functions independently of RhTRS1 to block PKR inhuman and rhesus cells, while RhTRS1 serves this function inAGM cells. Such an arrangement would be analogous to the VVsystem, in which E3L is critical for replication in many cell typesbut not in BHK cells, while K3L is required for efficient PKR an-tagonism in BHK cells but few others (27). Construction and anal-ysis of a RhCMV with deletion of its TRS1 gene will be importantfor distinguishing among these possibilities.

Regardless of how RhCMV is able to block PKR in order toreplicate in human and rhesus cells, our studies of the effects ofRhTRS1 in AGM cells reveal a new mechanism by which a CMVdsRNA binding protein can block the PKR pathway. Most signif-icantly, unlike HuTRS1, RhTRS1 does not block PKR autophos-phorylation but does still block the next step in the PKR pathway,the phosphorylation of eIF2�. It can thereby prevent the shutoff ofprotein synthesis and allow viral replication. HuTRS1 andRhTRS1 appear to have different PKR binding properties.HuTRS1 binds to inactive human PKR and inhibits its activation.HuTRS1 can also bind to phosphorylated human PKR. On theother hand, RhTRS1 binds to AGM PKR only after it has beenphosphorylated (Fig. 4 and 7). Thus, TRS1 proteins may differ intheir recognition of higher-order structural determinants, such assurfaces created or exposed by the conformational changes thatPKR undergoes as a result of binding to dsRNA, dimerization, andautophosphorylation (12). It may be that RhTRS1 binding to ac-tivated AGM PKR interferes with the conformational change nec-essary to reposition serine 51 of eIF2� during the kinase reaction(13).

RhTRS1 is not the only viral protein that blocks the PKR kinaseafter the autophosphorylation step. The VV K3L gene encodes aPKR pseudosubstrate that mimics eIF2� and binds preferentiallyto activated PKR, preventing phosphorylation of eIF2� (12). Re-spiratory syncytial virus infection appears to allow PKR activationbut blunts its eIF2� kinase activity, possibly as a result of the viralN protein binding to PKR and shifting its association from eIF2�to protein phosphatase 2A (18). Although the effects of blockingPKR’s eIF2� kinase activity before or after autophosphorylationare similar with respect to translational initiation, it is possiblethat there are different consequences of the two mechanisms. Forexample, antagonist-bound activated PKR might still be able tophosphorylate other potential substrates (22), although addi-

tional studies will be needed to determine whether the viral antag-onists block these kinase reactions too.

Our studies reveal substantial differences in TRS1-PKR inter-actions and mechanisms in primates. It therefore seems paradox-ical that the PKR antagonists from a much more distantly relatedvirus, MCMV, function in human cells and that HuTRS1 canfunction in mouse cells (10, 44). It may be that under some assayconditions, such as overexpression resulting from transient trans-fection (10), the dsRNA binding properties of these antagonistsare sufficient to block the PKR pathway (and possibly otherdsRNA-activated pathways) and allow viral replication. Such amechanism may explain an N-terminal deletion mutant of E3Lthat binds to dsRNA but not to PKR and enables viral replicationin HeLa cells (39). However, this mutant fails to prevent eIF2�phosphorylation or protein synthesis inhibition at late times afterinfection in cell culture, fails to rescue pathogenesis in mice, and isunable to antagonize PKR in a yeast assay (26, 35). Another pos-sibility is that during adaptation to some of the many changesarising in PKR during Old World monkey evolution, RhTRS1 lostits ability to interact with surfaces of ancestral PKR alleles thatwere maintained in both the hominoid and rodent lineages. Fur-ther genetic and structural dissection of the interactions of PKRalleles and their CMV antagonists will be needed to resolve thispuzzle and to identify the specific settings in which PKR antago-nism contributes to the species specificity of CMV replication.

ACKNOWLEDGMENTS

We thank Bertram Jacobs (Arizona State University), Michael Axthelmand Klaus Früh (Oregon Health and Sciences University and Vaccine andGene Therapy Institute), Peter Barry (University of California, Davis),Michael Mathews (University of Medicine and Dentistry of New Jersey),and Denise Galloway (FHCRC) for reagents and Harmit Malik (FHCRC)and Nels Elde (FHCRC) for reagents and helpful discussions. We alsothank Krystal Fontaine (University of Washington) and the GenomicsCore of the FHCRC for technical assistance.

This work was supported by NIH AI027762 (to A.P.G.) and K08AI067138 (to G.B.).

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Species-Specific PKR Antagonism by CMV TRS1 Genes

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Species Specificity of Protein Kinase R Antagonism byCytomegalovirus TRS1 Genes

Stephanie J. Child, Greg Brennan, Jacquelyn E. Braggin, and Adam P. Geballe

Divisions of Human Biology and Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA, and Departments of Microbiology and Medicine,University of Washington, Seattle, Washington, USA

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