Murine Cytomegalovirus m142 and m143 Are both Required To Block Protein Kinase R-Mediated Shutdown of Protein Synthesis

Cytomegaloviruses carry the US22 family of genes, which havecommon sequence motifs but diverse functions. Only two of the12 US22 family genes of murine cytomegalovirus (MCMV) are essentialfor virus replication, but their functions have remained unknown.In the present study, we deleted the essential US22 family genes,m142 and m143, from the MCMV genome and propagated the mutantviruses on complementing cells. The m142 and the m143 deletionmutants were both unable to replicate in noncomplementing cellsat low and high multiplicities of infection. In cells infectedwith the deletion mutants, viral immediate-early and early proteinswere expressed, but viral DNA replication and synthesis of thelate-gene product glycoprotein B were inhibited, even thoughmRNAs of late genes were present. Global protein synthesis wasimpaired in these cells, which correlated with phosphorylationof the double-stranded RNA-dependent protein kinase R (PKR)and its target protein, the eukaryotic translation initiationfactor 2 ${alpha}$ , suggesting that m142 and m143 are necessary to blockthe PKR-mediated shutdown of protein synthesis. Replicationof the m142 and m143 knockout mutants was partially restoredby expression of the human cytomegalovirus TRS1 gene, a knowndouble-stranded-RNA-binding protein that inhibits PKR activation.These results indicate that m142 and m143 are both requiredfor inhibition of the PKR-mediated host antiviral response.

INTRODUCTION

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Introduction

Cytomegaloviruses (CMVs) are prototypes of the ß subfamilyof the Herpesviridae. Their genomes span 230 kb and are thelargest among the herpesviruses. Of the approximately 170 geneson a CMV genome, about 46 are conserved among the herpesviruses(42). An additional 34 genes are characteristic of the ß-herpesviruses(12, 21, 44, 58), and the remaining genes are unique to a particularvirus.

A typical property of ß-herpesvirus genomes is thepresence of gene families, which probably arose by duplicationfrom ancestral genes. One of the largest, the US22 gene family,was first identified in human cytomegalovirus (HCMV) and waslater also found in other ß-herpesviruses (12, 21,44, 51, 58). It is characterized by four conserved sequencemotifs consisting of hydrophobic residues interspersed withcharged amino acids. HCMV and murine cytomegalovirus (MCMV)both include 12 members of the US22 gene family on their genomes(12, 51). The rat CMV and human herpesviruses 6 and 7, two otherhuman ß-herpesviruses, also possess up to 11 US22family genes (20, 21, 44, 58).

Little is known about the functions of US22 gene products. However,5 of the 12 US22 gene products of MCMV affect the virus' abilityto replicate in macrophages: M36, M43, m139, m140, and m141(28, 29, 32, 41). The molecular mechanism of action for onlyone of these has been elucidated. The M36 gene encodes an antiapoptoticprotein that binds to procaspase 8 and inhibits death receptor-mediatedinduction of apoptosis (41). The positional and sequence homologof M36 in HCMV, UL36, was originally proposed to function asa transcriptional transactivator (16). However, a later studyshowed that UL36 encodes a viral inhibitor of caspase 8 activation(vICA), a function it shares with its counterpart in MCMV (40,41, 54).

A comprehensive mutational analysis of all 12 US22 gene familymembers of MCMV identified m142 and m143 as the only two genesof this family that are essential for virus replication in cellculture (41). These two genes are transcribed with immediate-earlykinetics and lack motif II, one of the four conserved motifs(26, 27). The functions of these two proteins, however, remainunknown. The assumption that immediate-early proteins are likelyto function as transcriptional transactivators—as hasbeen shown for the HCMV TRS1 and IRS1 gene products (52, 55)—couldnot be confirmed for m142 and m143 (26).

In the present study, we investigated the functions of m142and m143 during MCMV infection. Viral deletion mutants of thesegenes replicated only on complementing cells and were unableto express viral late proteins and to amplify viral DNA in normalfibroblasts. They failed to inhibit activation of the double-strandedRNA (dsRNA)-dependent protein kinase R (PKR) and shutdown ofprotein synthesis, suggesting that this antiviral response preventsreplication of the deletion mutants. Consistent with our results,Child et al. report that m142 and m143 form a dsRNA-bindingcomplex and that the two proteins can jointly rescue a vacciniavirus deficient for the dsRNA-binding protein E3L (13).

MATERIALS AND METHODS

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Materials and Methods

Plasmids and genes. The TRS1, m142, and m143 genes were amplified by PCR from theHCMV AD169 and the MCMV genomes, respectively, using the followingprimers: 5'-A AAG AAT TCC ACC ATG GCC CAG CGC AAC GGC ATG TCG-3'(TRS1-fw), 5'-AAA CTC GAG TCA AGC GTA GTC TGG GAC GTC GTA TGGGTA TTG AGC ATT GTA ATG GTA GT-3' (TRS1-rev), 5'-A AAG AAT TCCACC ATG GAC GCC CTG TGC GCG GC-3' (m142-fw), 5'-AAA CTC GAGTCA AGC GTA GTC TGG GAC GTC GTA TGG GTA GTC GTC ATC GTC GGCGTC CGC-3' (m142-rev), 5'-AAA GGA TCC ACC ATG TCT TGG GTG ACCGGA GAT-3' (m143-fw), and 5'-A AAG AAT TCA AGC GTA GTC TGG GACGTC GTA TGG GTA CGC GTC GGT CGC TCT CTC GTC-3' (m143-rev). Suitablerestriction sites (in italics) for cloning in pcDNA3 (Invitrogen)and a hemagglutinin (HA) epitope sequence (underlined) at the3' end were introduced by the primers. It is noteworthy thatthe m143 gene sequence differed from the published sequence(51) by the presence of an additional guanosine residue betweenpositions 201402 and 201403 of the MCMV sequence (GenBank accessionnumber NC_004065). This leads to a frameshift compared to thepublished sequence analysis and a termination of the m143 openreading frame (ORF) at nucleotide position 200963 of the MCMVgenome. The same difference was independently detected by others(13), suggesting that the published sequence is incorrect.

Retroviral transduction. The genes of interest were excised from pcDNA3 and insertedinto the retroviral vector plasmid pLXSN (TRS1 and m142) orpLXRN (m143). Production of Moloney murine leukemia virus-basedretroviral vectors using the Phoenix packaging cell line andtransduction of NIH 3T3 cells was done as in a previous study(8). Transduced cells were selected with 700 µg/ml G418and grown as bulk cultures without clonal selection.

Mutagenesis. All mutant viruses were constructed on the basis of an MCMVvariant expressing the green fluorescent protein (MCMV-GFP),which has been used in previous studies (7, 8, 31). The MCMV-GFPgenome, cloned as a bacterial artificial chromosome (BAC), wasmodified in the Escherichia coli strain DY380 as described previously(8, 9). For the construction of the deletion mutants, a zeocinresistance gene (zeo) was PCR amplified with primers that contained50-nucleotide (nt) sequences homologous to positions 199621to 199670 and 200749 to 200798 (for ${Delta}$ m142) or 201121 to 201170and 202594 to 202643 (for ${Delta}$ m143). For construction of the m142revertant genome, the plasmid pBSo142 was constructed, whichis based on pBluescriptII KS(+) (Stratagene) and contains asynthetic oligonucleotide encoding 50-nt sequences up- and downstreamof the m142 ORF, spaced by EcoRI, EcoRV, XhoI, and HpaI restrictionsites. The HA-tagged m142 gene and a kanamycin resistance gene(kan) were inserted into pBSo142 using the EcoRI/XhoI and HpaIsites, respectively. The entire cassette was excised with SacIand ApaI and used for repair of the m142 deletion. The kan gene(which was flanked by FRT sites) was subsequently removed usingFLP recognition target (FLP) recombinase as described previously(8). The m143 revertant genome was constructed by the same strategywith plasmid pBSo143, containing the 50-nt homologies up- anddownstream of the m143 ORF. To replace m142 and m143 with TRS1,we inserted the TRS1 gene into pBSo142 and pBSo143, respectively,as had been done for the revertants. Alternatively, TRS1 wasalso inserted at an ectopic position, replacing the nonessentialORFs m02 to m06. This was done using the pReplacer plasmid asdescribed previously (31). Wild-type and recombinant MCMV BACDNAs were isolated from E. coli using NucleoBond PC100 columns(Macherey-Nagel). The viral genomes were digested with suitablerestriction endonucleases and separated on 0.6% agarose gels.The DNA was subsequently transferred to a nylon membrane usinga TurboBlotter device (Schleicher & Schuell). Hybridizationwith a digoxigenin-labeled probe directed against the m142 andm143 genes and chemiluminescent detection were performed usinga DIG-High Prime DNA-labeling and detection kit (Roche) accordingto the manufacturer's recommendations.

Cells and viruses. MCMV was propagated on NIH 3T3 cells (ATCC CRL-1658) accordingto standard procedures (6). To reconstitute recombinant virusesfrom mutant genomes, BAC DNA was transfected into normal orcomplementing NIH 3T3 cells. Virus stocks were produced on thesame cells and titered using the 50% tissue culture infectiousdose (TCID₅₀) method (39). For growth kinetics, cells were seededin six-well plates and infected with MCMV at the indicated MOI.Two hours after infection, the cells were washed with phosphate-bufferedsaline, and fresh medium was added. The medium was replacedat the indicated time points, and the virus content in the supernatantwas determined by titration. All growth kinetics experimentswere done in triplicate. To analyze viral-DNA replication, totalDNA was extracted from infected NIH 3T3 cells, blotted ontoa nylon membrane, and detected with a digoxigenin-labeled probeagainst the MCMV M45 gene as described above.

Protein detection. For Western blot analysis, cells were lysed in RIPA buffer containing20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% Na deoxycholate, 1%Triton X-100, 0.1% sodium dodecyl sulfate, and a protease inhibitorcocktail (Roche). Denatured protein samples were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis andtransferred to nitrocellulose membranes (Amersham). For immunologicaldetection, the following antibodies were used: CROMA101 againstMCMV IE1 and CROMA103 against E1 (both provided by Stipan Jonjic,University of Rijeka, Rijeka, Croatia), and 2E8.21A againstthe MCMV envelope glycoprotein B (gB) and 3B9.22A against M44(both provided by Lambert Loh, University of Saskatchewan, Saskatchewan,Canada). Monoclonal antibodies against the HA epitope tag (16B12[Covance Research Products] and 3F10 [Roche]), ß-actin(A5316; Sigma), and murine PKR (B-10; Santa Cruz) and polyclonalrabbit antibodies against phosphorylated and total murine eIF2 ${alpha}$ (the ${alpha}$ subunit of eukaryotic translation initiation factor 2;Cell Signaling) were purchased from suppliers as indicated.Horseradish peroxidase-coupled secondary antibodies (Dako andCell Signaling) and enhanced chemiluminescence reagents (Amersham)were used to develop the blots. For immunofluorescence, cellswere grown on coverslips, fixed with 3% paraformaldehyde, andpermeabilized with 0.3% Triton X-100. Proteins were detectedusing an anti-HA primary antibody and an Alexa Fluor 488- orAlexa Fluor 594-coupled secondary antibody (Molecular Probes).Nuclei were stained with propidium iodide or 4',6-diamidino-2-phenylindoledihydrochloride (DAPI). Fluorescent images were generated witha Zeiss LSM 510 confocal microscope.

For metabolic labeling, NIH 3T3 cells were infected at a multiplicityof infection (MOI) of 5 TCID₅₀/cell. Twenty-four hours afterinfection, the cells were incubated for 1 h in medium containing[³⁵S]methionine and [³⁵S]cysteine (143 µCi total radioactivity).Protein lysates of labeled cells were separated by polyacrylamidegel electrophoresis. The protein concentration for each lysatewas determined using a Bicinchoninic Acid Protein Assay kit(Pierce), and equal amounts of total protein were loaded ontoeach lane. The gel was fixed, dried, and exposed to an X-rayfilm (Kodak).

Real-time reverse transcription (RT)-PCR. Total RNA was isolated with Trizol reagent (Invitrogen), purifiedwith an RNeasy kit (QIAGEN), and subsequently digested withDNAse I to remove contaminating DNA. The quality of the RNApreparations was confirmed by denaturing agarose gel electrophoresis.First-strand cDNA synthesis was performed with Superscript IIIreverse transcriptase (Invitrogen) using 125 ng random-hexamer-primedtotal RNA. The reverse-transcribed total RNA was digested withRNAse H. For each sample, a background control without the additionof reverse transcriptase was made.

Real-time PCR analysis was performed using a Lightcycler 1 instrument(Roche). Each reaction mixture was made with the SYBR GreenFastStart Kit (Roche) in 15 µl total volume containing2.5 mM MgCl₂, 7.5 pmol of each primer, and 1 ng reverse-transcribedtotal RNA. As a housekeeping gene control, 18S rRNA was chosen,because previous experiments had shown that its level of expressionis not significantly affected by MCMV infection. Each run wasperformed with 45 cycles, followed by a melting-curve analysisto confirm the identities of the PCR products. For the detectionof viral- and housekeeping gene transcripts, the following primerswere used: 5'-TTA TGG TTC CTT TGG TCG CTC G-3' (18s-fwd), 5'-CACCGG GTT GGT TTT GAT CTG A-3' (18s-rev), 5'-GCG ATG TCC GAG TGTGTC AAG-3' (gB-fwd), 5'-CGA CCA GCG GTC TCG AAT AAC-3' (gB-rev),5'-TCG TTC GTG AAC ATC GTG GTG-3' (gM-fwd), 5'-GAT CGC GTT GTACAT CGT CAG G-3' (gM-rev), 5'-TGC ACC AGG CGC TCT GTA AC-3'(M44-fwd), and 5'-CGC TGA GGA AGT TCT CGA TGG-3' (M44-rev).Relative quantification was carried out according to the methodof Pfaffl (48).

RESULTS

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Results

Construction of MCMV m142 and m143 deletion mutants. To analyze the functions of m142 and m143 in the context ofan MCMV infection, we constructed deletion mutants of thesegenes. MCMV-GFP was used as a basis for the construction ofmutant viruses, because GFP expression by infected cells facilitatesthe monitoring of potentially growth-defective viruses.

A BAC clone of MCMV-GFP was modified by homologous recombinationin E. coli. The m142 and m143 ORFs were replaced individuallywith a zeocin resistance gene (Fig. 1A). In a second step, revertantgenomes were constructed by replacing the zeocin resistancegene of the deletion mutants with an HA epitope-tagged m142or m143 gene, respectively (Fig. 1A). A kanamycin resistancegene flanked by FRT sites was inserted downstream of the respectivegene to serve as a selectable marker for homologous recombination.This cassette was subsequently removed by FLP recombinase, leavingonly a single FRT site behind. The restriction patterns of thesegenomes showed the expected changes (Fig. 1B). Transfectionof the wild-type and the revertant genomes into NIH 3T3 fibroblastsyielded replication-competent virus, but the ${Delta}$ m142 and ${Delta}$ m143 deletionmutants did not grow. Individual green fluorescent cells wereobtained after transfection of the genomes, but these cellsdisappeared after repeated passaging of the cells.

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FIG. 1. Construction of MCMV deletion mutants. (A) The m142 and m143 open reading frames were replaced by a bacterial zeocin resistance gene (zeo), using recombinant BAC technology. To obtain the revertants, Rm142 and Rm143, the HA-tagged open reading frames, together with an FRT-flanked kanamycin resistance gene, were inserted into the MCMV ${Delta}$ m142 and ${Delta}$ m143 genomes, respectively. The kan gene was subsequently removed using FLP recombinase, leaving a single FRT site (black oval) behind. EcoRI (E) restriction sites and the expected fragment sizes are indicated. wt, wild type. (B) EcoRI-digested MCMV BACs were separated on an ethidium bromide-stained agarose gel. The arrowheads indicate altered fragments. (C) Southern blot of EcoRI-digested viral DNA from MCMV-infected cells hybridized with m142- and m143-specific probes, respectively.

To overcome this problem, NIH 3T3 cells were transduced withretroviral vectors carrying m142 or m143, respectively, anda G418 resistance gene for selection of the transduced cells.As shown in Fig. 2A and B, the cells expressed the HA-taggedproteins. They could be detected by Western blotting and immunofluorescence.When these complementing cells were transfected with the genomesof the MCMV deletion mutants, the mutant viruses could be reconstituted.To confirm the identities of the recombinant viruses, viralDNA was extracted from virus preparations and analyzed by Southernblot hybridization. All mutants showed the expected band patterns(Fig. 1C). Moreover, the revertant viruses expressed HA-taggedm142 or m143 proteins, respectively (Fig. 2C and D).

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FIG. 2. Expression of m142 and m143 in transduced and infected cells. (A) Western blot and (B) indirect immunofluorescence analysis of NIH 3T3 cells stably transduced with retroviruses encoding HA-tagged m142 and m143 proteins, respectively. (C) Detection of HA-tagged m142 and m143 proteins expressed by the revertant MCMVs Rm142 and Rm143, respectively, by Western blotting and (D) immunofluorescent staining of infected NIH 3T3 cells. Nuclei were stained with propidium iodide (PI) or DAPI. mock, mock infected.

Growth properties of the ${Delta}$ m142 and ${Delta}$ m143 deletion mutants. It is conceivable that transfection of an MCMV genome into fibroblastsprovides less favorable conditions for initiation of the viralreplication cycle than an infection. Hence, the failure to regeneratea virus from a mutant BAC, as shown in a previous study (41),provided only indirect evidence for essential roles of the m142and m143 genes. Moreover, it is possible that a gene deletionprevents virus replication during an infection at a low MOIbut that this defect can be overcome by infecting cells at ahigh MOI. This has been shown, for instance, for the HCMV IE1gene, which is required for efficient growth at a low MOI butis dispensable for replication at a high MOI (22, 43). Therefore,we infected murine fibroblasts at low and high MOIs and analyzedvirus replication. The growth curves in Fig. 3A and B show thatthe ${Delta}$ m142 and ${Delta}$ m143 viruses did not grow on noncomplementing fibroblasts,either at a low or at a high MOI. However, the viruses grewto almost normal titers on complementing cells (Fig. 3C and D).This indicated that each of the two genes is essential for virusgrowth in cell culture at both low and high MOIs.

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FIG. 3. Growth kinetics of recombinant MCMVs. Replication of the ${Delta}$ m142 and ${Delta}$ m143 deletion mutants and revertants in NIH 3T3 cells was analyzed upon infection at (A) a low or (B) a high MOI. (C) Replication of the ${Delta}$ m142 mutant in m142-expressing NIH 3T3 cells and (D) of the ${Delta}$ m143 mutant in m143-expressing cells. The error bars represent the standard errors.

m142 and m143 are required for late-protein synthesis and DNA replication. To identify a block in the cascade of viral-gene expressionas a potential reason for the inability of the deletion mutantsto replicate, we analyzed the expression of immediate-early( ${alpha}$ ), early (ß₁), early-late (ß₂), and late( ${gamma}$ ) MCMV proteins by Western blotting. To exclude the possibilitythat gene expression by the mutant viruses is delayed, we analyzedthe expression of these proteins not only at 24, but also at72 h postinfection (p.i.). Although expression of the ${alpha}$ geneIE1 (34) was reduced in ${Delta}$ m142- and ${Delta}$ m143-infected cells, the levelsof immediate-early proteins were apparently sufficient to activateexpression of the ß₁ gene E1 (10) and the ß₂gene M44 (38), as their levels were not dramatically alteredcompared to the wild type and the revertants. A major differencewas seen in the levels of the late protein gB (37, 50), whichwas barely detectable in cells infected with the deletion mutants(Fig. 4A and B). Moreover, viral DNA was present in comparableamounts in these cells at 24 h p.i., but an amplification tohigh levels at very late times (72 h p.i.) apparently did notoccur (Fig. 4C and D).

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FIG. 4. Viral-gene expression and DNA replication. NIH 3T3 cells were infected with recombinant MCMVs at an MOI of 0.5 TCID₅₀/cell. Viral-gene products were detected with specific antibodies 24 (A) and 72 (B) h p.i. Detection of viral-DNA amplification in infected cells by slot blotting and hybridization with an MCMV-specific probe 24 (C) and 72 (D) h p.i. ßa, beta actin; wt, wild type; mock, mock infected.

m142 and m143 are required to prevent PKR activation and shutdown of protein synthesis. Earlier studies identified a transcriptional transactivatingfunction of the HCMV US22 gene family proteins TRS1, IRS1, andUL36 in assays using transient transfection of expression andreporter plasmids (16, 52, 55). Although m142 and m143 failedto show similar transactivating activities in a more recentstudy (26), we wanted to test whether m142 and m143 are requiredfor late-gene transcription. The mRNA levels of the early-lategene M44 and the late genes M55/gB and M100/gM (36) were measuredby real-time PCR. At 24 h p.i., the mRNA levels of these geneswere not significantly altered in the deletion mutants comparedto wild-type or revertant viruses (Fig. 5A). Only at very latetimes (72 h p.i.) were the mRNA levels reduced about 10-foldin cells infected with ${Delta}$ m143 (Fig. 5B). This suggested that thedefect in late-protein expression is based primarily on a posttranscriptionalblock. Indeed, when global protein synthesis was analyzed bymetabolic labeling with [³⁵S]methionine and [³⁵S]cysteine, itwas found to be markedly reduced in cells infected with the ${Delta}$ m142 or ${Delta}$ m143 mutant (Fig. 6A).

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FIG. 5. Real-time RT-PCR quantification of MCMV late-gene transcripts. NIH 3T3 cells were infected at an MOI of 1 TCID₅₀/cell with wild-type (wt) and mutant MCMV. Total RNA was harvested (A) 24 and (B) 72 h p.i. and quantified by real-time RT-PCR. Two RNA preparations from cells infected with different virus stocks were made, and from each preparation, two to four independent quantitative PCRs per sample were done. The transcript abundance in MCMV wt-infected cells was defined as 1. The abundances of transcripts from mutant viruses are shown relative to the wt. The error bars represent the standard errors. mock, mock infected.

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FIG. 6. Shutdown of protein synthesis in ${Delta}$ m142- and ${Delta}$ m143-infected cells. (A) NIH 3T3 cells were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine 24 h p.i. at an MOI of 5 TCID₅₀/cell. Cell lysates (10 µg total protein per sample) were separated by gel electrophoresis and analyzed by autoradiography. (B) Detection of phosphorylated PKR and eIF2 ${alpha}$ (P-eIF2 ${alpha}$ ) in ${Delta}$ m142- and ${Delta}$ m143-infected cells by Western blotting. Total eIF2 ${alpha}$ and ß-actin were used as controls. mock, mock infected; wt, wild type.

Global shutdown of protein synthesis is a well-known innatedefense mechanism by which infected cells can inhibit the productionof progeny virus (47, 53). This process usually involves activationof the double-stranded-RNA-dependent PKR and PKR-mediated phosphorylationof eIF2 ${alpha}$ , an essential cofactor for translation of mRNAs intopolypeptides. When PKR and eIF2 ${alpha}$ were analyzed in infected cellsby Western blotting, a more slowly migrating form of PKR wasdetected only in cells infected by the deletion mutants, butnot in cells infected with the wild-type or the revertant virus(Fig. 6B). The more slowly migrating band most probably representsphosphorylated (i.e., activated) PKR, because treatment of NIH3T3 cells with poly(I · C), a known inducer of PKR phosphorylation,resulted in a comparable band shift (data not shown). Unfortunately,an antibody specifically recognizing phosphorylated murine PKRwas not available, and thus, the identity of the more slowlymigrating band could not be formally proven. However, phosphorylationof the PKR target protein eIF2 ${alpha}$ was clearly detected in thesecells (Fig. 6B), suggesting that the shutdown of protein synthesisis indeed mediated by PKR-dependent phosphorylation of eIF2 ${alpha}$ .

HCMV TRS1 can restore the growth of ${Delta}$ m142 and ${Delta}$ m143. The TRS1 and IRS1 proteins of HCMV have recently been shownto rescue a vaccinia virus (VV) lacking the dsRNA-binding proteinE3L (14, 15). Similarly, TRS1 and IRS1 were able to reversethe PKR-mediated protein synthesis shutoff induced by a recombinantherpes simplex virus type 1 (HSV-1) lacking the ${gamma}$ ₁34.5 gene (11).In addition, TRS1 was shown to possess dsRNA-binding activity(24). These results suggested that TRS1 and IRS1 are responsiblefor inhibiting a shutdown of protein synthesis during an HCMVinfection, although this has not yet been formally demonstrated.

TRS1 and IRS1 share a number of properties with m142 and m143of MCMV. They are expressed at immediate-early times, are membersof the US22 gene family, and lack one of the four sequence motifscharacteristic of this gene family (27). However, the sequencesimilarity of TRS1 to m142 and m143 is noticeable, but not striking.The m143 protein has 18% identical and 30% similar amino acidsto both m142 and TRS1. The m142 protein shares only 11% identityand 21% similarity with TRS1 (Fig. 7). Hence, we asked if TRS1could rescue the growth defect of the MCMV ${Delta}$ m142 and/or ${Delta}$ m143virus. To investigate this, we infected NIH 3T3 cells stablytransduced with a TRS1-expressing retrovirus and studied thegrowth of the ${Delta}$ m142 and ${Delta}$ m143 deletion mutants. Both mutants grewon TRS1-expressing fibroblasts (Fig. 8A), albeit to 10- to 100-fold-lowertiters than the wild-type virus. We also inserted the TRS1 geneinto the genomes of the ${Delta}$ m142 and ${Delta}$ m143 mutants in order to testif TRS1 expression in cis would also rescue virus growth. Surprisingly,insertion of TRS1 in place of the deleted m142 and m143 genes(Fig. 8B) did not rescue virus growth (data not shown). However,when TRS1 was driven by a phosphoglycerate kinase gene (pgk)promoter and inserted at an ectopic position, replacing thenonessential genes m02 to m06 (Fig. 8C), virus growth on NIH3T3 fibroblasts and expression of the late protein gB were restored(Fig. 8D to F). These results indicated that TRS1 can, at leastin part, compensate for a lack of m142 or m143. Since TRS1 canblock the PKR-dependent antiviral response during an HSV-1 orVV infection (11, 14), the results also support the conceptthat m142 and m143 are both required to prevent PKR-mediatedshutdown of protein synthesis.

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FIG. 7. Alignment of the m142, m143, and TRS1 proteins. Amino acid residues conserved between the three proteins are indicated by black boxes; similar residues are marked with open boxes. The alignment was generated with Multalign software (18). The TRS1 sequence was truncated at both ends, and only the overlapping part is shown. US22 gene family motifs I, III, and IV are marked. Periods represent gaps in gene sequences and nonconserved residues in the consensus sequence.

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FIG. 8. TRS1 rescues m142 and m143 deletion mutants. (A) Multistep growth kinetics of the wild type (wt) and deletion mutants on NIH 3T3 cells stably transduced with a TRS1-expressing retroviral vector. (B) Introduction of the HA-tagged TRS1 gene into the MCMV genome, either by replacing the deleted gene or (C) by insertion at a distant position together with a pgk promoter. (D) Multistep growth curves of the T ${Delta}$ m142 and T ${Delta}$ m143 mutants on NIH 3T3 fibroblasts. (E) Viral-protein expression 24 and (F) 72 h p.i. at an MOI of 0.5 TCID₅₀/cell. The error bars represent the standard errors.

DISCUSSION

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Discussion

Replication of intracellular pathogens, such as viruses, canbe inhibited by innate immune defenses of the host cell. Oneof the best characterized is the interferon-mediated antiviralresponse, which proceeds in three phases (reviewed in reference25). First, the cell senses the presence of a virus via toll-likereceptors (TLRs) that recognize pathogen-associated molecularpatterns (33). TLR signaling triggers the expression and secretionof beta interferon (IFN-ß). Binding of IFN-ßto type I interferon receptors on the same and on neighboringcells activates the Jak/STAT signaling pathway, which leadsto an upregulation of antiviral effector molecules, such asPKR, 2'-5'-oligoadenylate synthetase, and Mx proteins. Viruses,on the other hand, have evolved various mechanisms to counteractthe IFN-mediated antiviral response on different levels (reviewedin reference 25).

CMVs already trigger the activation of the IFN response duringattachment and entry into the cell. Contact of gB with its receptoractivates the IFN-responsive pathway via interferon regulatoryfactor 3 (3, 4). CMV also activates inflammatory-cytokine productionby engaging the pattern recognition receptors TLR2, TLR9, andCD14 (17, 35). However, HCMV counteracts the induction of IFN-ßexpression via its tegument protein pp65/UL83 (1, 5) and/orvia a combined action of the neighboring gene product, pp71/UL82,and the IE2 protein, as suggested by later studies (56, 57).IFN receptor signaling is also inhibited by both HCMV and MCMV.The HCMV IE1 protein forms a complex with the signal transducerand activator of transcription 1 (STAT1) and STAT2 and inhibitsthe induction of IFN-responsive effector genes (46). In MCMV,the M27 gene product inhibits both type I and type II IFN signalingby selectively down-regulating STAT2 without affecting STAT1(60).

Like many other viruses, HCMV also encodes proteins that binddsRNA and inhibit the dsRNA-dependent activation of PKR. Thisfunction is fulfilled by the TRS1 and IRS1 proteins of HCMV(11, 14, 24). These two proteins are identical over the N-terminaltwo-thirds of their amino acid sequences, because they are locatedin part in the short repeat region of the HCMV genome (12).Each of the two proteins can rescue a VV E3L or an HSV-1 ${gamma}$ ₁34.5deletion mutant, suggesting at least partly redundant functions.Unfortunately, the importance of these two proteins for thelife cycle of HCMV could not yet be analyzed, because a TRS1-IRS1double-knockout mutant could not be grown in normal fibroblasts,and the generation of reliable complementing cells proved tobe difficult (W. Brune, J. E. Adamo, and T. Shenk, Abstr. 27thInt. Herpesvirus Workshop, abstr. 11.13, 2002, and W. Brune,unpublished results).

The present study shows that m142 and m143 are both requiredto inhibit PKR activation and a shutdown of protein synthesisat late times postinfection. The inability of the ${Delta}$ m142 and ${Delta}$ m143mutants to prevent PKR activation is by itself not sufficientto prove that the two proteins are directly involved in blockingPKR activation. However, the fact that TRS1, a known inhibitorof PKR activation, can rescue the ${Delta}$ m142 and ${Delta}$ m143 deletion mutantssuggests a direct role of m142 and m143 in this process. Hence,this is the first study that demonstrates the importance ofthe PKR-mediated antiviral response during a cytomegalovirusinfection.

The MCMV m142 and m143 proteins show significant sequence similarityto their presumed counterparts in HCMV, TRS1 and IRS1 (Fig.7). They differ, however, from TRS1 and IRS1 in that each ofthem is essential for MCMV replication. The HCMV proteins, bycontrast, are together essential only for HCMV replication (Bruneet al., Abstr. 27th Int. Herpesvirus Workshop), consistent withredundant functions of the two proteins. These findings suggesteither that the m142 and m143 proteins block different stepstoward PKR activation or that they form an inhibitory complex.The latter possibility is supported by a report which showsthat m142 and m143 can bind dsRNA and rescue a VV deficientfor the dsRNA-binding protein E3L only when expressed together(13).

Inhibition of the dsRNA-dependent antiviral response is obviouslya crucial task for RNA viruses, as their replication inevitablyresults in the generation of dsRNA molecules. However, a recentstudy showed that DNA viruses, such as herpesviruses, vacciniavirus, or adenoviruses, also produce dsRNA (59). One possibleexplanation is that DNA viruses carry genes on both strandsof their genomes. This can lead to the formation of dsRNA byhybridization of (partly) complementary mRNAs transcribed fromopposing strands. Another possibility is that these virusesneed to prevent PKR activation through viral microRNAs. Recentstudies have shown that HCMV, like many other herpesviruses,have such microRNAs (19, 23, 49).

The essential roles of m142, m143, and TRS1/IRS1 may not relyexclusively on their abilities to block the PKR-mediated antiviralresponse. Many herpesvirus proteins are multifunctional, andthis may also hold true for the proteins studied here. For instance,previous studies have demonstrated that TRS1 or IRS1 is requiredfor transient complementation of HCMV DNA replication (30, 45).This is consistent with our observation that MCMV DNA is notreplicated to high levels in the absence of m142 or m143 (Fig.4). However, it is possible that MCMV DNA replication is notblocked completely but only inhibited at late times postinfectionas an indirect consequence of the antiviral response. In fact,the detection of late-gene transcripts argues for at least limitedviral-DNA replication, as ${gamma}$ -gene transcription should occur onlyafter DNA replication.

A recent study has revealed that TRS1 (but not IRS1) is requiredfor efficient assembly of DNA-containing capsids (2), and thisfunction is apparently not secondary to a reduced transcriptionor translation of viral genes. The molecular mechanism for thisadditional function of TRS1 has not yet been determined, noris it known whether m142, m143, or any other MCMV protein servesa similar function.

Considering the multiple functions of the HCMV TRS1 protein,it may appear surprising that TRS1 rescued gB expression andreplication in the ${Delta}$ m142 and ${Delta}$ m143 mutants only partly. Insertionof the TRS1 gene into the genomes of the deletion mutants restoredvirus replication only when TRS1 expression was driven by anextraneous pgk promoter. Since the endogenous promoters of m142and m143 are known to be weak during the initial phase of infection(26), artificial overexpression of TRS1 appears to be requiredto compensate for the lack of m142 or m143. This could indicatethat these MCMV and HCMV proteins are not fully capable of blockingthe antiviral response in cells of a heterologous host species.

The genes of the US22 family probably arose from a common ancestralgene but have diverged in their specific functions. However,it is remarkable that all of the US22 family genes for whicha function has been determined are involved in the subversionof innate or adaptive immune functions. At least in some cases,the functions were conserved between the human and the murinecytomegaloviruses: UL36 and M36 inhibit tumor necrosis factoralpha- and Fas-mediated signaling by blocking caspase 8 activation(41, 54), and TRS1/IRS1 and m142/m143 inhibit the IFN-dependentantiviral response by preventing PKR activation (references11, 13, 14, and 24 and this study). Thus, it should not be surprisingif other US22 family genes, whose functions are as yet unknown,are also engaged in immune evasion.

ACKNOWLEDGMENTS

We thank Katharina Wolf for valuable help with confocal microscopyand Stipan Jonjic and Lambert Loh for providing antibodies.We also thank Adam Geballe for communicating unpublished resultsand Michael Nevels for critical reading of the manuscript.

This work was supported by the Emmy Noether Program of the DFG(Br 1730/2-3), SFB 421, and the Georg und Agnes Blumenthal Stiftung.

FOOTNOTES

* Corresponding author. Mailing address: Robert Koch-Institut, Fachgebiet Virale Infektionen, Nordufer 20, 13353 Berlin, Germany. Phone: 49 30 18754 2502. Fax: 49 1810754 2502. E-mail: brunew{at}rki.de.

Present address: Universitätsklinik Mannheim, Institutfür Medizinische Mikrobiologie und Hygiene, Abteilung fürVirologie, 68167 Mannheim, Germany.

REFERENCES

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