Estrogen-related receptor α is required for efficienthuman cytomegalovirus replicationJesse Hwanga,1, John G. Purdyb, Kai Wua, Joshua D. Rabinowitzb, and Thomas Shenka,2

Departments of aMolecular Biology and bChemistry and the Lewis-Sigler Institute for Integrative Genomics Princeton University, Princeton, NJ 08544

Contributed by Thomas Shenk, November 24, 2014 (sent for review October 21, 2014)

An shRNA-mediated screen of the 48 human nuclear receptorgenes identified multiple candidates likely to influence the pro-duction of human cytomegalovirus in cultured human fibroblasts,including the estrogen-related receptor α (ERRα), an orphan nu-clear receptor. The 50-kDa receptor and a 76-kDa variant wereinduced posttranscriptionally following infection. Genetic andpharmacological suppression of the receptor reduced viral RNA,protein, and DNA accumulation, as well as the yield of infectiousprogeny. In addition, RNAs encoding multiple metabolic enzymes,including enzymes sponsoring glycolysis (enolase 1, triosephos-phate isomerase 1, and hexokinase 2), were reduced when thefunction of ERRα was inhibited in infected cells. Consistent withthe effect on RNAs, a substantial number of metabolites, whichare normally induced by infection, were either not increased orwere increased to a reduced extent in the absence of normal ERRαactivity. We conclude that ERRα is needed for the efficient produc-tion of cytomegalovirus progeny, and we propose that the nuclearreceptor contributes importantly to the induction of a metabolicenvironment that supports optimal cytomegalovirus replication.

herpesvirus | metabolism | nuclear receptor | mass spectrometry

Human cytomegalovirus (HCMV) belongs to the β-herpesvirussubfamily, and although most healthy individuals remain

asymptomatic subsequent to infection, the pathogen is a majorcontributor to birth defects and to life-threatening disease inimmunocompromised patients (1–3). As with all viruses, HCMVdepends on the host cell to provide macromolecular buildingblocks for virion production, and throughout the course of itsevolution, HCMV has adapted to manipulate numerous funda-mental cellular processes, including RNA accumulation (4),translation (5), metabolism (6, 7), secretory pathways (8), and thecell cycle (9).The nuclear receptor gene family, of which there are 48

members in the human genome, encodes transcription factors(10). Some bind to specific hydrophobic ligands such as steroids,vitamin D, fatty acids, and lipids, providing a means to sensechanges in the extracellular or intracellular environment; others are“orphans” with no known ligand, and are often constitutively active(11). As transcription factors that modulate broad gene regulatorynetworks, nuclear receptors control numerous physiological pro-cesses, including aspects of metabolism (cell autonomously and atthe whole-organism level), development, and cellular differentia-tion, cancer, circadian rhythm, and immunity (11).The first indication that nuclear receptor signaling modulates

HCMV replication surfaced after the identification of a retinoicacid response element within the major immediate-early pro-moter (MIEP) that caused a dose-dependent response to retinoicacid in reporter assays (12). Retinoic acid was further shown toenhance viral DNA replication and progeny production in humanembryonal carcinoma cells and foreskin fibroblasts (13, 14). Theligand also exacerbated symptoms of infection, which were re-versed by retinoic acid receptor antagonists, in a mouse modelusing murine CMV (15), arguing that the vitamin A metaboliteplays a physiologically relevant role in CMV infection.Two additional nuclear receptors reported to affect HCMV

replication are the glucocorticoid receptor and the peroxisome

proliferator-activated receptor γ (PPARγ). Cortisol, the gluco-corticoid receptor ligand, enhanced virus production in humanembryonic kidney cells (16). Most recently, a PPAR responseelement was described within the MIEP, and a PPARγ antago-nist reduced viral protein expression, lipid droplet formation,and virus production (17).To replicate efficiently, HCMV induces numerous metabolic

and lipidomic changes (18, 19), and the intimate connectionbetween metabolism and nuclear receptors (20, 21) offersa compelling reason to examine the role of these transcriptionfactors during infection more closely. In addition, nuclear recep-tors are also known to modulate inflammatory signaling (22)and cell cycle progression (23), pathways that are important forsuccessful infection.To explore potential roles for additional nuclear receptors in

the HCMV replication cycle, we performed an shRNA screen inpermissive primary fibroblasts to identify host factors thatmodulate virus production. Multiple hits were identified, and theorphan nuclear receptor, estrogen-related receptor α (ERRα),was further validated and characterized to be essential for opti-mal replication of both laboratory and clinically derived strainsof HCMV.

ResultsNuclear Receptor shRNA Screen Identifies Putative Regulators ofHCMV Replication. Each of the 48 human nuclear receptor geneswas targeted by using two independent shRNAs, and their effecton HCMV yield was quantified (Fig. 1A). Nuclear receptor-specific shRNAs and a luciferase-targeting control shRNA weredelivered to MRC5 fibroblasts using a lentivirus vector (pLKO)that also expressed a puromycin resistance gene (24). At 24 hposttransduction, fresh medium was applied with puromycin

Significance

Viruses use the host cell’s resources, including numerous cel-lular metabolites, to successfully replicate and produce prog-eny. Here, we report that estrogen-related receptor α (ERRα),an orphan nuclear receptor and transcriptional regulator, sup-ports the expression of multiple metabolic enzymes thatcontribute to the metabolic program induced by human cyto-megalovirus in cultured primary fibroblasts. Loss or inhibitionof ERRα impedes the viral replication cycle at multiple levels,leading to a reduction in progeny virus. Our findings identifyERRα as a host factor that is manipulated by the virus for itsreplicative needs and establish its potential as a pharmacolog-ical target for treatment of cytomegalovirus infection.

Author contributions: J.H., J.G.P., K.W., J.D.R., and T.S. designed research; J.H., J.G.P., andK.W. performed research; J.H., J.G.P., K.W., J.D.R., and T.S. analyzed data; and J.H., J.G.P.,K.W., J.D.R., and T.S. wrote the paper.

The authors declare no conflict of interest.1Present address: Section of Infectious Diseases, Department of Internal Medicine, YaleUniversity, New Haven, CT 06520.

2To whom correspondence should be addressed. Email: tshenk@princeton.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422361112/-/DCSupplemental.

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(1 μg/mL) to select for cells containing an shRNA, and drugselection was maintained for a total of 96 h. To monitor trans-duction efficiency, analysis with a GFP-expressing lentivirusperformed in parallel demonstrated that >99% of transducedpuromycin-selected cells expressed the marker.At 6 d after the transduction process was initiated, fibroblasts

were infected at a multiplicity of 0.3 50% tissue culture infectivedose (TCID50) per cell in the absence of puromycin with a phe-notypically wild-type derivative of HCMV, ADinUL99-GFP,expressing GFP fused to the pUL99 late viral protein. At 96 hpostinfection (hpi) culture supernatants were assayed for in-fectious virus, and the yield was determined relative to thatobtained from luciferase shRNA-expressing cells. The dataset(Dataset S1) was then normalized by robust z score, whichcompares each shRNA relative to the distribution of the entirescreen and is insensitive to outliers (25), and plotted in ascendingorder (Fig. 1B). A negative z score indicates that knockdown ofa nuclear receptor reduced HCMV yield, and a positive scoreindicates that knockdown increased yield. When the z-scorefrequency was plotted (Fig. 1C), a near-normal distribution wasobserved. The threshold for designating a nuclear receptor asa hit was set as those targets for which a single shRNA achievedan absolute robust z score ≥2.3, which is 10-fold greater than therobust z score of control shRNA (0.23). This ranking identifiedeight nuclear receptors that might support HCMV replication incultured fibroblasts (negative z scores) (Table 1).Because each score represents results obtained for a single

shRNA, it is important to recognize that hits in the screen do notunambiguously identify nuclear receptors that modulate HCMVyield; rather, they are candidates for further validation. Deepercoverage of the screen using additional shRNA sequences wouldlikely result in multiple hits targeting the same gene and help torule out false positives. In addition, the screen does not rule outa role for nuclear receptors that do not meet the cut off. How-ever, PPARγ, which is already known for its positive role duringHCMV infection (17), was classified as a hit in the screen, sug-gesting our methodology was robust and may identify other hostfactors that play a role during viral replication.Among the hits, we focused initially on ERRα, a constitutively

active orphan nuclear receptor with no known endogenous ligand

or estrogen binding activity (26). Our choice of ERRα was basedon its role in cellular metabolism. ERRα is a transcriptionalregulator of cellular metabolic networks (26), and it can con-tribute to a cancer-like metabolic profile (27). HCMV infectionalso modulates glycolysis, the TCA cycle and fatty acid metabo-lism (18, 28), and it induces a cancer cell-like metabolic signature(6, 18). Consequently, it seemed possible that ERRα activitymight underlie aspects of HCMV metabolic manipulations.

Confirmation of a Role for ERRα During HCMV Infection. To validatea role for ERRα during HCMV replication, the shRNA sequenceused to generate the hit in the screen (shRNA1) plus an additionalshRNA construct targeting ERRα (shRNA2) were evaluatedfor their ability to reduce ERRα levels at various times after in-fection with the AD169 strain of HCMV (ADwt) (Fig. 2A, Left).Compared with control shRNA, both ERRα-targeting shRNAsreduced the level of ERRα, and shRNA1 almost completely ab-rogated expression of the nuclear receptor. Whereas shRNA1 didnot exhibit cytotoxicity in uninfected fibroblasts, as assayed bytrypan blue exclusion assay (>95% viable), shRNA2 displayedlimited toxicity (85–90% viable). ERRα knockdown reduced theproduction of ADwt progeny (Fig. 2A, Right). Fibroblasts trans-duced with lentivirus expressing shRNA1 or -2 produced ∼300-fold and 60-fold less virus, respectively, compared with controlshRNA-expressing cells at 96 hpi. The magnitude of reduction invirus yield correlated with the extent of ERRα suppression.

Posttranscriptional Regulation of ERRα Isoforms Following HCMVInfection. To evaluate the effect of HCMV on ERRα mRNAand protein expression, time course experiments were carriedout following ADwt infection or mock infection of fibroblasts.qRT-PCR analysis showed that the amount of ERRα mRNA didnot change at 24 and 48 hpi and was elevated to a limited extent(∼40%) relative to mock-infected cells at 72 hpi (Fig. 2B).However, the amount of ERRα protein increased beginning at48 hpi and was markedly elevated at 72 hpi (Fig. 2C), showingthat the nuclear receptor is substantially up-regulated by a post-transcriptional mechanism following HCMV infection.When ERRα was assayed by Western blot, two species were

evident: a band migrating at the known 50-kDa size of ERRα plusa 76-kDa doublet moiety that was present only at late times afterinfection (Fig. 2C). To ensure that the antibody did not spuriouslyrecognize a nonspecific band, samples were immunoblotted withanti-ERRα antibody in the presence or absence of a 150-foldexcess of the peptide used as immunogen to produce the anti-body. The addition of the peptide abolished reactivity with boththe 50-kDa and 76-kDa bands (Fig. 2D), arguing that the largerspecies is a modified isoform of ERRα.The accumulation of ERRα protein after 24 hpi temporally

coincides with the onset of viral DNA accumulation, which isrequired for late viral protein synthesis (29). To examine if viral

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Fig. 1. Nuclear receptor shRNA screen. (A) Design and timeline of theshRNA screen. MRC-5 cells were transduced with lentiviruses expressing anshRNA plus a puromycin (Puro)-resistance gene; cells were selected with Puro(1 μg/mL) and infected with ADinUL99-GFP (0.3 TCID50 per cell). Infectiousvirus in culture medium was quantified at 96 hpi. (B) Robust z-score valuesfor individual shRNAs. Dotted lines indicate the cutoff value of ±2.3. (C)Frequency distribution of robust z scores from the screen.

Table 1. Nuclear receptors identified as hits in the shRNA screen

Nuclear receptor name Gene ID Z score

NR1I2 (PXR, pregnane X receptor) 8856 −8.74ESR2 (ERβ, estrogen receptor β) 2100 −6.88ESRRG (ERRγ, estrogen-related receptor γ) 2104 −5.03HNF4G (HNF4γ, hepatocyte nuclear factor 4 γ) 3174 −3.70NR2C1 (TR2) 7181 −3.40ESRRA (ERRα, estrogen-related receptor α) 2101 −3.11RXRG (RXRγ, retinoid x receptor γ) 6258 −2.69PPARG (peroxisome proliferator-activated

receptor γ)5468 −2.45

Genes for which one shRNA generated an absolute robust z score ≥ 2.3 wereidentified as hits in the screen. A complete list of robust z scores is provided inDataset S1.

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DNA synthesis and subsequent late protein production are re-quired for the posttranscriptional up-regulation of ERRα, cul-tures were treated with phosphonoacetic acid (PAA), an inhibitorof the viral DNA polymerase (30), or solvent alone as a control.As expected, PAA prevented accumulation of a late viral protein,pUL99, without affecting the level of the immediate-early IE1protein (Fig. 2E). The level of the 50-kDa ERRα isoform did notchange, but accumulation of the 76-kDa isoform was dramaticallydiminished in response to the drug. Thus, the two ERRα isoformsexhibit differential dependence on viral DNA synthesis and theproduction of late viral proteins.

Pharmacological Inhibition of ERRα Is Detrimental to HCMV Replication.Although ERRα is an orphan nuclear receptor with no knownendogenous ligand, the drug XCT790 acts as an inverse agonist tocounteract the constitutive activity of ERRα (31). To complementthe analysis using shRNA knockdown of ERRα, the effect ofXCT790 on ADwt production was investigated. In addition toinhibiting the transcriptional activity of ERRα, XCT790 causes itsproteasomal degradation (32). Treatment with the compound re-duced the protein levels of both ERRα species, and the addition ofproteasome inhibitor MG-132 prevented the degradation (Fig. 3A,Left). The compound induced a dose-dependent reduction in virusyield, resulting in a 25-fold decrease at 96 hpi in the presence of2.5 μM of XCT790 (Fig. 3A, Right).

ADwt is a laboratory strain of HCMV that is restricted toreplication in fibroblasts. To determine the importance of ERRαduring infection of a different cell type with a clinical isolate ofHCMV, TB40/E-GFP (28, 33), was used to infect retinal pig-mented epithelial cells (ARPE-19) (34). TB40/E spreads in acell-to-cell manner without release of significant quantities ofextracellular infectious particles in these cells (35). shRNAknockdown of ERRα (Fig. 3B, Left) and antagonism withXCT790 (Fig. 3B, Right) both led to a marked decrease in plaquesize, when assayed by counting the number of IE1-positive cellsper visible plaque. Thus, ERRα supports the production of cell-free virus in fibroblasts by a laboratory strain and direct cell-to-cell spread in epithelial cells by a clinical isolate of HCMV.

Localization of ERRα During Viral Replication. ERRα is normallylocalized to the nucleus, and unlike many nuclear receptors, itdoes not require ligand binding in the cytoplasm for trans-location to the nucleus (36). To determine whether infectionalters its normal localization pattern, ADwt-infected and un-infected fibroblasts were examined by indirect immunofluores-cence assay, using the viral IE2 protein as a nuclear marker forinfected cells (Fig. 4A). The change in nuclear morphology ofinfected cells is indicative of dynein-induced nuclear shapechanges that are characteristic of HCMV infection (37). From 8 to48 hpi, ERRα remained in the nucleus. However, at 72 hpi, ERRαwas present in both the nucleus and cytoplasm (Fig. 4A), tempo-rally correlating with the appearance of the 76-kDa isoform of thenuclear receptor (Fig. 2 C–E). Nuclear-cytoplasmic fractionationof infected cells at 72 hpi showed that the 76-kDa ERRα isoformis present exclusively in the cytoplasm, whereas the 50-kDa bandwas present in both fractions (Fig. 4B). Additional bands reactingwith the ERRα antibody, not seen in other immunoblots, may bedegradation products of the 76-kDa species generated duringcell fractionation.

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Fig. 2. ERRα supports the production of HCMV progeny and is inducedposttranscriptionally. (A) shRNAs targeting ERRα reduced the yield of HCMV.Fibroblasts were transduced with control (C) or ERRα-targeting shRNA (1 or2) and subsequently infected with HCMV AD169wt (3 TCID50 per cell). ERRαwas monitored by immunoblot assay at the indicated times (Left), and in-fectivity (infectious units, IU) in the supernatant was titered at 96 hpi (Right).(B) ERRα RNA is induced to a limited extent by infection. Fibroblasts wereinfected with AD169wt (3 TCID50 per cell) or mock infected, and RNA sam-ples were harvested for qRT-PCR analysis at the indicated times. AU, arbi-trary units. (C) ERRα protein is substantially induced during infection.Fibroblasts were infected or mock infected as in B, and protein samples wereanalyzed by immunoblot assay. Immediate-early 1 protein (IE1) and pUL99are HCMV proteins. (D) Specificity of higher molecular weight band (***) ofERRα. Fibroblasts were infected as in B, and the immunogenic peptide usedto generate the antibody (molar ratio of antibody to peptide, 1:150) blockedrecognition of the 50- and 76-kDa ERRα isoforms. (E) Viral DNA replication isrequired for induction of higher molecular weight isoform, but not lowermolecular weight isoform, of ERRα. Similar to B, except cells were treatedwith phosphonoacetic acid (PAA, 100 μg/mL) or ethanol solvent control.

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Fig. 3. Pharmacological inhibition of ERRα reduces the yield of HCMV. (A)XCT790 reduces the levels of ERRα proteins within infected cells (Left).Fibroblasts were infected with AD169wt (3 TCID50 per cell), and indicatedsamples were treated with MG-132 (5 μM) at 43 hpi, before harvesting at 49hpi for immunoblot analysis. XCT790 (2.5 μM) reduced the yield of infectiousvirus (IU, infectious units) at 96 hpi (Right). Control cultures that did notreceive drug were treated with the solvent, DMSO. (B) Genetic and phar-macological inhibition of ERRα reduced the yield of the clinical HCMV iso-late, TB40/E-GFP, in epithelial cells. ARPE-19 cells were transduced withcontrol or ERRα shRNA-1, infected with TB40/E-GFP (0.1 TCID50 per cell), andthe number of immediate-early 1 protein (IE1)-positive nuclei per plaquewas quantified by immunofluorescence at 8.5 dpi. For drug treatment, ARPE-19 cells were similarly infected, treated with indicated concentrations ofXCT790 or DMSO, and immunostained for IE1-positive nuclei at 8.5 dpi.Three biological replicates were performed. Error bars represent ±1 SD.

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Late during infection, HCMV induces the formation of a cy-toplasmic organelle called the assembly compartment (AC) atwhich numerous viral and cellular proteins accumulate (38), in-cluding the HCMV late protein, pUL99. ERRα did not colocalizewith pUL99 (Fig. 4C), arguing that it does not preferentially re-side in the assembly compartment. The drug PAA, which blocksHCMV DNA replication and the synthesis of late viral proteins,prevented the accumulation of cytoplasmic ERRα (Fig. 4D),consistent with its ability to block accumulation of the 76-kDaisoform (Fig. 2E) and confirming that the large isoform is inducedby a late viral function.

ERRα Is Required for the Efficient Accumulation of Viral Proteins andDNA. To probe further into the role of ERRα during viral repli-cation, a protein accumulation time course experiment was per-formed comparing ERRα-deficient and control cells (Fig. 5A,Left). Fibroblasts expressing relevant shRNA constructs wereinfected with ADwt or mock infected. ERRα normally functionsby interacting with PGC-1 family coactivators (39). The levelof PGC-1α protein increased following infection, and ERRαknockdown had no effect on its accumulation. Infection did notdetectably influence the level of PGC-1β. Several virus-codedproteins were also monitored. The accumulation of the immedi-ate-early protein IE1 and an early protein pUL38 was not influ-enced by ERRα levels. However, the amount of IE2 at 48 and72 hpi was considerably reduced upon ERRα knockdown, eventhough at 24 hpi, there was no difference between the knockdownand control cells. Although IE2 is an immediate-early viral pro-tein, it accumulates to an even greater level as the infection pro-ceeds (40). In addition, there was a delay in the expression of thepUL26 and pUL44 early proteins and the late pUL99 protein.The decrease in early and late protein expression suggested

that there might be a defect in viral DNA synthesis. This turned

out to be the case, as ERRα knockdown caused a reduction inthe accumulation of viral DNA throughout the course of theinfection; at 72 hpi, knockdown cells contained ∼25-fold lessviral DNA than control cells (Fig. 5A, Right).XCT790 treatment affected viral protein and DNA accumu-

lation in a similar manner as observed in the shRNA knockdownexperiment, including pUL69, a viral protein expressed withearly-late kinetics (41) (Fig. 5B). Finally, a second drug, AM251,which is an endocannabinoid receptor antagonist that displaysadditional inhibitory activity toward ERRα and causes its pro-teasomal degradation (42), also generated a similar protein ex-pression profile (Fig. 5C).To delve further into how ERRα depletion influenced viral

gene expression, a PCR array targeting 32 HCMV genes acrossall temporal expression classes was used (Fig. 6 and Table S1).Similar to the viral protein expression pattern, at 24 hpi there waslittle difference in viral mRNA accumulation between ERRα andcontrol knockdown cells. Although we detected late transcripts insamples harvested during the early stage of viral replication, theylikely resulted from incoming RNA packaged into virions (43).Differences in transcript levels were evident at later times afterinfection. Most viral RNAs assayed at these times were present atreduced levels in ERRα-deficient conditions; and UL82, UL83,and UL111A RNAs were reduced to the greatest extent, showingapproximately 5- to 10-fold reductions at 72 hpi. These resultssuggest that the lack of ERRα obstructs the normal progressionof the HCMV gene expression program at the level of mRNAaccumulation, beginning at some point following 24 hpi.

ERRα Knockdown Reduces the Levels of Multiple RNAs EncodingProteins Involved in Central Carbon Metabolism. ERRα is a con-stitutively active transcription factor that can regulate numer-ous genes involved in glycolysis and TCA cycle (26, 27, 36).

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Fig. 4. A portion of ERRα is relocalized from the nucleus to cytoplasm during viral replication. (A) Immunofluorescence assay. Fibroblasts were infected withAD169wt (0.1 TCID50 per cell) and processed for immunofluorescence assay at indicated time points. The immediate-early 2 protein (IE2) was monitored asa nuclear viral marker. (B) Nuclear-cytoplasmic fractionation of infected cells. Fibroblasts were infected with AD169wt (3 TCID50 per cell), and at 72 hpi,nuclear and cytoplasmic extracts were separated. PARP1 and tubulin are controls for nuclear and cytoplasmic proteins, respectively. (C) Cytoplasmic ERRα doesnot colocalize with the viral assembly compartment protein, pUL99. Fibroblasts were infected with ADinUL99-GFP (0.1 TCID50 per cell), fixed at 72 hpi, andprocessed for immunofluorescence assay. (D) ERRα remains nuclear in the absence of viral DNA replication. Fibroblasts were infected with AD169wt (0.5TCID50 per cell) and treated with phosphonoacetic acid (PAA) or ethanol. Cells were analyzed as before at 72 hpi. ***, modified isoform.

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Hence, ERRα may play a similar role during HCMV infection,contributing to metabolic alterations induced by the virus. TargetedPCR arrays were used to determine if glucose and mitochondrialmetabolic gene expression is altered in HCMV-infected cells whenERRα expression is repressed (Fig. 7A). Cells expressing ERRαshRNA or a control shRNA were infected with ADwt at a mul-tiplicity of 3 TCID50 per cell and RNAs were analyzed 48 h later.RNAs corresponding to multiple genes that encode for mito-chondrial proteins were down-regulated by at least a factor of twofollowing knockdown of ERRα, including metaxin 2 (MTX2),solute carrier family 25 A4 (SLC25A4), and A25 (SLC25A25),and uncoupling protein 1 (Ucp1). Similarly, multiple glycolyticRNAs were down-regulated, such as those encoding enolase 1

(ENO1), triosephosphate isomerase 1 (TPI1), and hexokinase 2(HK2). Amylo-1,6-glucosidase,4-alpha-glucanotransferase RNA,whose product is required for glycogen breakdown and mobi-lization of glucose (44), was also down-regulated. The onlyRNAs in the PCR arrays that were up-regulated at least two-fold upon ERRα knockdown encoded translocase of outermitochondrial membrane 40 (TOMM40) and Bcl2-binding com-ponent 3 (BBC3).PCR array hits were validated by performing qRT-PCR

analysis on RNA samples prepared at various times after in-fection (Fig. 7B). All of the above hits were confirmed, exceptfor TOMM40, which showed a slight reduction in expressioninstead of up-regulation as indicated by the PCR array. ATP-citrate lyase (ACLY) showed only a minor reduction uponERRα knockdown, consistent with the failure to detecta change in level of this RNA in the PCR array. ERRα has beenreported to regulate the cell-surface epidermal growth factorreceptor (EGFR) in certain cell lines (42), but ERRα knock-down had no effect on EGFR during HCMV infection (Fig.7B). In sum, our data demonstrate that ERRα is required ininfected cells for maximal expression of multiple RNAsencoding proteins directly involved in central carbon andmitochondrial metabolism.

Steady-State Levels of Central Carbon Metabolites and NucleotidesWithin Infected Cells Are Reduced upon ERRα Knockdown. Thedecrease in expression of multiple metabolic genes followingknockdown of ERRα suggested that it might contribute to themetabolic perturbations accompanying HCMV infection. Toinvestigate this possibility, a liquid chromatography-mass spec-trometry (LC-MS)-based metabolomics experiment was per-formed. Cells expressing ERRα shRNA or control shRNA weremock infected or infected with ADwt at a multiplicity of 3TCID50 per cell. In addition, HCMV-infected or mock-infectedcells were treated with XCT790 or DMSO. At 48 hpi, sampleswere harvested and analyzed.Increased levels of glycolytic and TCA cycle intermediates were

evident when infected cells were compared with mock-infectedcells (Fig. 8A, columns 1 and 3), as has been observed previ-ously (28, 45). When the levels of metabolites in infected cellsexpressing ERRα shRNA or treated with XCT790 were comparedwith infected cells in which the nuclear receptor was not knockeddown or pharmacologically inhibited (Fig. 8A, columns 2 and 4),many of the HCMV-mediated increases in metabolite levels wereblunted, i.e., the profiles for many metabolites following infection

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Fig. 5. Inhibition of ERRα function suppresses viral protein and DNA accu-mulation. (A) Analysis of viral proteins and DNA following shRNA-mediatedknockdown of ERRα. Fibroblasts were transduced with control or ERRα shRNA-1,infected with AD169wt (3 TCID50 per cell) or mock infected, and proteinsamples were immunoblotted at the indicated times (Left). IE1, IE2, pUL26,pUL38, pUL44, and pUL99 are HCMV proteins. For viral DNA (vDNA)analysis, total DNA from infected cells was extracted and viral DNA wasquantified by using qPCR (Right). (B) Analysis of viral proteins and DNAfollowing inhibition of ERRα with XCT790. Fibroblasts were infected withAD169wt (3 TCID50 per cell) and treated with XCT790 (2.5 μM) or DMSO.Protein samples and viral DNA were analyzed as in A. (C ) Analysis of viralproteins and DNA following inhibition of ERRα with AM251. Same as in B,except AM251 (4 μM) or DMSO were applied to infected cells. ***, modi-fied isoform. Three biological replicates were performed. Error bars rep-resent ±1 SD.

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IE DE LL L TL UN

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A (s

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Con

trol)

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Fig. 6. Knockdown of ERRα broadly reduces the steady-state levels of viralRNAs. Fibroblasts were transduced with control or ERRα shRNA-1, infectedwith AD169wt (3 TCID50 per cell), and processed for PCR array targeting viralgenes of all temporal classes. Viral genes are categorized as immediate early(IE), early (E), leaky late (LL), late (L), true late (TL), or unknown kinetics (UN).Two biological replicates were performed and each was analyzed in dupli-cate. Error bars represent ±1 SD.

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versus ERRα inhibition were negatively correlated (Fig. 8B).Importantly, as the glucose metabolism targeting PCR array datapredicted (Fig. 7), we observed a reduction in multiple glycolyticmetabolites, including hexose phosphate, 2,3-bisphosphoglycerate,fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and phos-phoenolpyruvate. Further, most ribonucleoside mono-, di-, andtriphosphates as well as deoxyribonucleoside mono-, di-, and tri-phosphates assayed were present at reduced levels within infectedcells following ERRα inhibition.The congruent results from genetic and pharmacological in-

hibition indicate that ERRα supports aspects of the metabolicinduction that occurs during viral replication.

DiscussionThe human genome encodes 48 functional nuclear receptors (10),which sense a multitude of ligands and respond to specific extra- orintracellular stimuli (46, 47). Several nuclear receptors scored in anshRNA screen for effects on the yield of HCMV (Fig. 1B andDataset S1), some with potential to inhibit and others that mightsupport efficient viral growth. Our screen produced several can-didates that require further validation; however, we validatedone hit, ERRα, by confirming its effect on HCMV protein ex-pression and yield following treatment with two independentshRNAs as well as two different pharmacological inhibitors(Figs. 2, 3, and 5).

B

ENO1 (-2.59) TPI1 (-2.09)

ACO2 (-2.03)

AGL (-3.22)

HK2 (-2.46)

MDH1B (-2.31)

Glucose Metabolism

Control shRNAERR shRNA

3.0

Nor

m. t

o P

PIA

(AU

)

2.0

0

3.0

0

3.0

0

8.0

0

6.0

4.0

1.0

2.0

1.0

2.0

1.0 2.0

ERR HK2 ANT1 AGL

15

10

0

3.0

0

3.0

0

5

2.0

1.0

2.0

1.0

ACO2 ENO1 MTX2 TPI13.0

0

2.0

1.0

15

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15

0

15

0

5

10

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10

5

SLC25A25 BBC3 TOMM40 ACLY6.0

0

4.0

2.0

24 48 72 24 48 72 hpi

24 48 72 24 48 72 hpi

2.0

1.5

0

6.0

0

1.04.0

2.0

UL21A EGFR

0.5

Nor

m. t

o P

PIA

(AU

) N

orm

. to

PP

IA (A

U)

Nor

m. t

o P

PIA

(AU

)

MTX2 (-3.3)

SLC25A4 (-3.6)

SLC25A25 (-2.2)

UCP1 (-7.7)

TOMM40 (3.3)

BBC3 (3.0)

Mitochondrial Metabolism

Log 1

0 (E

RR

shR

NA

2-C

t )

A

Log10 (Control shRNA 2- Ct)

Glucose Metabolism

Fig. 7. ERRα knockdown reduces expression of multiple RNAs encoding metabolic enzymes in infected cells. (A) qRT-PCR analysis of arrays that interrogate RNAsrelated to glucose metabolism and mitochondrial function. ERRα shRNA or control shRNA expressing fibroblasts were infected with AD169wt (3 TCID50 per cell) andprocessed 48 h later by using qPCR arrays. Numbers in parentheses indicate fold reduction (green) or induction (red) in ERRα knockdown cells. Dotted lines representtwofold changes. (B) Confirmation of PCR array hits. Fibroblasts were transduced and infected as in A, but RNA samples were harvested at 24, 48, and 72 hpi. Fullgene names are provided in Table S2. Values were normalized to cellular PPIA RNA. Four biological replicates were performed. Error bars represent ±1 SD.

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ERRα protein is induced substantially by HCMV infection.The change is posttranscriptional, because ERRα RNA levelsincrease to only a limited extent (Fig. 2B). Posttranscriptionalinduction of ERRα has also been shown to occur upon T-cellactivation (48). ERRα from mock-infected cells migrates asa well-characterized 50-kDa species, but much of the increasefollowing infection is due to the accumulation of a 76-kDamoiety (Figs. 2, 3, 4, and 5). Three lines of evidence argue thatthe large species is a bona fide ERRα isoform. First, the peptidethat was used to generate the antibody blocked detection of thelarge species in a Western blot assay (Fig. 2D). Second, shRNA

knockdown reduced the level of the high molecular weight iso-form (Fig. 5A). Third, both XCT790 and AM251 caused deg-radation of the 76-kDa isoform (Fig. 5 B and C). As ERRα isknown to be SUMO modified (49), the larger species may rep-resent SUMOylated ERRα, although the shift in apparent mo-lecular weight (∼26 kDa) of the isoforms is greater than the15–17 kDa predicted for this modification (50). Perhaps ERRαundergoes multiple posttranslational modifications or a splicevariant is produced in HCMV-infected cells. Accumulation ofthe 76-kDa variant required viral DNA and late protein synthesis(Fig. 2E); and, whereas the 50-kDa ERRα species localized to

-3 0 3 log2

Citrate/IsocitratedAMPIndoleacrylic acid dTMPcyclic-AMP Oxaloacetate Myo-inositol DeoxyguanosineAminoadipic acid 2-dehydro-D-gluconate 2-keto-isovalerate dTTPOctulose bisphosphateGlutamate dATPRibose-phosphate 5-phosphoribosyl-1-pyrophosphate Sedoheptulose phosphate dUTPdGMPFumarateMalate N-acetyl-glutamate dCTPGlycerateDihydroxyacetone phosphate CTP dTDPXanthosine-5-phosphate UTP UDP dGTP4-Pyridoxic acid 2,3-bisphosphoglycerate Hexose phosphate D-gluconate

Aconitate3-phosphoserine Pyrophosphate Adenosine 5-phosphosulfate 3-methylphenylacetic acid N-acetyl-glucosamine-1,6-phosphate Pyruvate sn-glycerol-3-phosphate GuanosineD-glucarateThymidine Octulose phosphate IndolePyroglutamic acid DeoxyadenosinePantothenateUracil Aspartate CMP Erythrulose-1-phosphate D-glyceraldehyde-3-phosphate L-arginino-succinate IMP UMP

-ketoglutarateSuccinate N-acetyl-L-alanine Sedoheptoluse bisphosphate6-phospho-D-gluconate GTP Fructose-1,6-bisphosphate 3-phosphoglycerate ATP GDP CDP XanthosinePhosphoenolpyruvateProlineGMP

-4

-3

-2

-1

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-2 0 2 4 6

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ock

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SO

)

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s)

AD

wt+

shR

NA

1/C

AD

wt/M

ock

(DM

SO

)

Adw

t+X

C79

0/D

Fig. 8. ERRα is required for HMCV-driven modulation of metabolome. (A) ERRα knockdown or inhibition alters the metabolic profile of HCMV-infected cells.Fibroblasts expressing ERRα shRNA-1 or control shRNA were infected with AD169wt (3 TCID50 per cell) or mock infected. For pharmacological inhibition,cultures were treated with XCT790 or DMSO at 2 hpi. Cells were harvested and metabolites analyzed at 48 hpi. Column 1: ADwt/Mock (shRNAs) = [ADwt +control shRNA (shRNAC)]/[mock + shRNAC]; column 2: ADwt + shRNA1/C = [ADwt + ERRα-specific shRNA (shRNA1)]/[ADwt + shRNAC]; column 3: ADwt/mock(DMSO) = [ADwt + DMSO]/[mock + DMSO]; column 4: ADwt + XC790/D = [ADwt + XC790]/[ADwt + DMSO]. (B) Negative correlation between metabolitelevels in fibroblasts treated with ERRα-specific shRNA (Left, R = −0.379, P < 0.001) or an ERRα antagonist, XCT790 (Right, R = −0.493, P < 0.001) versus ADwt-infected fibroblasts. Each point represents a metabolite shown in A.

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both nucleus and cytoplasm during the late phase of HCMVinfection, the 76-kDa isoform was restricted to the cytoplasm ofinfected cells (Fig. 4B). To the best of our knowledge, granularcytoplasmic staining of endogenous ERRα has been shownpreviously only in a study of poorly differentiated breast cancertissue (51). Alternatively, the granular ERRα in the cytoplasmmight represent vesicular structures and reflect localization tothe late endocytic pathway. Because ERRα-dependent tran-scriptional activity is required for viral replication, the host cellmay sequester ERRα in the cytoplasm in an attempt to limitinfection. Even though nuclear receptors are most notable foraltering gene expression by directly binding to target DNAsequences, additional functions have been described (52, 53). Assuch, it is also conceivable that cytoplasmic ERRα binds hostand/or viral proteins to regulate aspects of infection.How does ERRα support HCMV replication? The steady-

state levels of several immediate-early viral proteins (IE1 andIE2) were not affected at 24 hpi by the loss of ERRα activity, butthe accumulation of two early (pUL26 and pUL44) and a lateviral protein (pUL99) was delayed and their levels were reduced(Fig. 5). IE2 RNA (Fig. 6) and protein levels (Fig. 5) were re-duced at 48 and 72 hpi, and this could in turn reduce the levels ofother viral gene products, because IE2 is a potent transcriptionalactivator that drives the cascade of HCMV gene expression (54).The reduced level of IE2 following knockdown or pharmaco-logical inhibition of ERRα argues that the nuclear receptor isrequired for its optimal expression during the late phase of thereplication cycle.How does ERRα support late IE2 expression? It is possible that

the nuclear receptor activates transcription of viral genes, includingthe major immediate-early promoter that is responsible for thesynthesis of mRNA encoding IE2. The timing of ERRα inductionfollowing infection (Fig. 2C) fits well with the reduction in IE2levels at 48 and 72 hpi in the absence of ERRα function (Fig. 5).ERRα could act directly by binding to the viral major immediate-early control region, although inspection of the sequence does notreveal a clear match to an estrogen-related receptor response el-ement. Alternatively, the nuclear receptor might not bind to theviral genome, but could act indirectly by modulating cellulartranscription factors that impact viral gene expression. Parenthet-ically, it is noteworthy that an in silico search for estrogen-relatedreceptor response elements in the viral genome identified a puta-tive binding site that conforms to a sequence known to conferERRα responsiveness (TCAAGGTCA) upstream of the UL21Atranscription start site. However, qRT-PCR analysis showed thatERRα had no effect on the level of UL21A RNA (Fig. 7B).ERRα supports aspects of metabolic changes induced by

HCMV infection, which may subsequently impact IE2 expres-sion. Although knockdown of ERRα did not completely reversethe metabolic phenotype induced by viral infection, the levels ofmany metabolites, including nucleoside mono-, di-, and tri-phosphates, were reduced (Fig. 8, columns 2 and 4), most likelyresulting in a suboptimal cellular environment for viral RNA andDNA synthesis, consistent with the observed reductions in ac-cumulation of viral DNA and RNAs (Figs. 5 A and B and 6).Late accumulation of IE2 RNA (and other viral transcripts)requires viral DNA replication (40), and this suggests that IE2accumulation during the late phase of the HCMV replicationcycle is obstructed when ERRα activity is reduced. This mayprevent a feed-forward mechanism where viral DNA replicationleads to even higher IE2 levels, which would then further stim-ulate expression of viral genes. Thus, insufficient ERRα activitylikely reduces viral RNA and protein expression indirectly, atleast in part by inhibiting viral DNA replication.Besides its impact on the synthesis of nucleic acids within

HCMV-infected cells by supporting the accumulation of nucleo-side and deoxynucleoside mono-, di-, and triphosphates, ERRα isgenerally required for cells and tissues with high energy demand

(55), and it is likely to impact viral replication and spreadthrough additional effects on metabolism. ERRα promotesa Warburg-like effect by inducing a metabolic shift from oxida-tive phosphorylation to glycolysis (27), and it has also beenshown to maintain the TCA cycle by inducing anaplerotic re-plenishment of TCA cycle metabolites (27). These changes arebroadly similar to alterations observed following HCMV in-fection of fibroblasts (6, 18, 28, 45), and knockdown of ERRαblunts many of these alterations following infection (Fig. 8),confirming a role for the nuclear receptor in the establishment ofthe viral metabolic program.How does ERRα contribute to the metabolic perturbations

induced by HCMV infection? PCR array analysis targetinga subset of genes encoding or regulating metabolic enzymesidentified several RNAs whose levels were decreased uponERRα knockdown (Fig. 7). Hexokinase 2 (HK2) and adeninenucleotide translocase 1 (ANT1) were among the hits, and bothare known to be regulated by ERRα (36, 56, 57). Both enzymessupport core metabolism: HK2 phosphorylates glucose (58) andANT1 exchanges mitochondrial ATP for cytosolic ADP (59).Additional candidate hits were identified in the nuclear re-

ceptor screen that might influence HCMV replication (Table 1).Of note, knockdown of pregnane X receptor (PXR) reducedvirus production. This nuclear receptor is involved in the de-toxification of xeno- and endobiotics, including cholesterol-derived compounds (60). HCMV infection increases cholesterolsynthesis (61) and intracellular cholesterol levels regulate theinfectivity of progeny virus (62), so PXR may be required toprevent accumulation of toxic cholesterol byproducts.In conclusion, ERRα contributes importantly to the altered

metabolic program induced by HCMV infection. When ERRαfunction is ablated, virus-induced metabolic changes are blunted,the cascade of viral gene expression is perturbed, and the pro-duction of infectious progeny is reduced. Our finding that anevolutionarily ancient orphan nuclear receptor is required for bothviral gene expression and metabolic perturbation suggests that thecoopting of this family of transcription factors may be a commonmechanism of viruses to replicate in their respective host niches.

MethodsCells, Viruses, and Reagents.HFFs (human foreskin fibroblasts) andMRC-5 cells(human embryonic lung fibroblasts) were cultured in DMEM supplementedwith 10% FBS and penicillin/streptomycin. For all experiments except for theinitial shRNA screen, cells were maintained in DMEM with 2% (vol/vol) di-alyzed FBS and penicillin/streptomycin.

A wild-type isolate of the HCMV AD169 strain (ADwt) was produced froma BAC clone by transfection of BAC plasmid (pAD/Cre) and pp71 expressionplasmid into HFFs to generate viral progeny of wild-type growth charac-teristics (63). ADinUL99-GFP, in which the tegument protein pUL99 is taggedwith GFP and exhibits wild-type growth kinetics, has been described (64).TB40/E-GFP (28), derived from an endotheliotropic clinical strain, TB40/E (65),contains a GFP expression cassette inserted between US34 and TRS1. All virusstocks were partially purified by centrifugation through a sorbitol cushion(20% sorbitol, 50 mM Tris·HCl,1 mM MgCl2, pH 7.2), concentrated 10-fold,and resuspended in DMEM supplemented with 3% (wt/vol) BSA. Viral stocktiters were determined by TCID50 on fibroblasts and stored at −80 °C untiluse. Infections were performed by treating cells with viral inoculum for 90min, followed by removal of inoculum and washing with serum-free DMEM.

Cell-free viral titers were determined by assaying for IE1-positive cells onreporter fibroblast plates. Infectious supernatant was applied to uninfectedfibroblasts, and 24–36 h later, cells were fixed with 4% (wt/vol) parafor-maldehyde and immunostained for IE1 (19, 66). For assaying cell-associatedviral spread, infected ARPE-19 cells were directly fixed with 4% (wt/vol)paraformaldehyde at 8.5 dpi and immunostained for IE1. The number of IE1-positive cells per plaque were counted as described previously (67).

Drugs were dissolved in DMSO (XCT790, Tocris; AM251, Cayman Chemicals;MG-132, Cayman Chemicals) or ethanol (PAA, Sigma-Aldrich), stored at −20degrees, and thawed once for application to cells.

Nuclear Receptor shRNA Screen. Lentiviral particles (pLKO.1) that target in-dividual nuclear receptor genes were purchased from Sigma-Aldrich (Dataset

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S1). Lentivirus expressing luciferase-specific shRNA and GFP-expressing len-tiviral particles were used as negative controls and to monitor the efficiencyof transduction, respectively. A total of 10,000 MRC-5 fibroblasts wereplated into each well (96-well plate); 18–20 h later, lentivirus inoculum (sixtransduction units per cell in 50 μL DMEM containing 10% (vol/vol) dialyzedFBS and 4 μg/mL polybrene) was added to each well, the 96-well plate wascentrifuged at 650 × g for 45 min at 25 °C, and placed in a 37 °C incubatorfor 24 h, after which the inoculum was removed and replaced with freshDMEM containing 10% (vol/vol) dialyzed FBS. After an additional 24 h,medium containing puromycin (1 μg/mL) was added for 96 h (the mediumwas changed and fresh drug added after 48 h). When the puromycin se-lection was completed, the transduced MRC-5 cells were infected withADinUL99-GFP at a multiplicity of 0.3 TCID50 per cell for 90 min and freshmedium was applied without puromycin. Medium was changed at 48 hpi,and supernatants were assayed for infectious virus at 96 hpi by using theOperetta High-Content Imaging system (Perkin-Elmer) to enumerate IE1-expressing nuclei (66). Values were normalized to the negative controlshRNA that was set to 1, log2 transformed, and robust z scores were calcu-lated (25).

Protein Analysis. To analyze proteins by immunoblot assay, cells were har-vested in buffer with protease inhibitor (Roche, EDTA-free tablets) andphosphatase inhibitors (1 mM sodium fluoride; 1 mM β-glycerophosphate;1 mM sodium orthovanadate; 10 mM sodium pyrophosphate), and analyzedas described previously (68). Nuclear-cytoplasmic extraction was performed asdescribed previously (69), except 350,000 cells were harvested. Primary anti-bodies to the following HCMV proteins were used: IE1 (clone 1B12) (70), IE2(clone 3A9) (71), pUL26 (clone 7H1-5) (68), pUL38 (clone 8D6) (72), pUL44(clone 10D8; Virusys), pUL83 (clone 8F5) (73), and pUL99 (clone 10B4-29) (74).Cellular proteins were assayed using antibodies to: ERRα (Novus), PGC-1α(Sigma-Aldrich), PGC-1β (Active Motif), PARP-1 (Cell Signaling), β-actin(Abcam), and tubulin (Sigma-Aldrich). Secondary antibodies were HRP-con-jugated goat anti-mouse and goat anti-rabbit IgG (Jackson ImmunoResearch).

Protein localization was analyzed by indirect immunofluorescence as de-scribed (35). Primary antibodies to ERRα and IE2 are described above; sec-ondary antibodies were goat anti-mouse or goat anti-rabbit conjugated withAlexa Fluor-488 or -555 (Life Technologies), and nuclei were counterstainedwith DAPI. Images were obtained by using a Nikon A1 confocal microscope.

Nucleic Acid Quantification. To quantify RNA levels using human mitochondria(PAHS-087ZA) and human glucose metabolism (PAHS-006ZA) PCR arrays (SABiosciences), total RNAwas extractedusing aDirect-zol RNA kit (ZymoResearch).Genomic DNA elimination and reverse transcription were done according to themanufacturer’s PCR array protocol. Triplicate biological samples were pooledsubsequent to reverse transcription for each experimental condition, and PCRarrays were processed according to the manufacturer’s protocol on an ABIPRISM 7900HT Sequence Detection System. Data analyses were performed us-ing the manufacturer’s software (www.sabiosciences.com/pcrarraydataanalysis.php). Results were normalized to GAPDH.

To confirm results of the PCR arrays for selected cellular RNAs, 200 ng oftotal RNA was reverse transcribed using qScript cDNA SuperMix (QuantaBiosciences) according to the manufacturer’s protocol. Real-time PCR wasperformed with SYBR Green PCR Mastermix (Applied Biosystems) usingindicated primers (Table S2) and values were normalized to cellularPPIA mRNA.

To quantify viral RNAs by using an HCMV qPCR array, primers weredesigned against the annotated AD169 strain (GenBank: FJ527563) usingQuantPrime (75) and checked by BLAST algorithm (blast.ncbi.nlm.nih.gov/Blast.cgi) to assure appropriateness for AD169-BAC (pAD/Cre, GenBank:AC146999) used in this study. Five cellular reference genes were included ascontrols (PPIA, PGK1, ACTB, HPRT1, and B2M). Primer sets (Table S2) werevalidated by: (i) standard curve analysis using serial dilutions of purifiedAD169 BAC DNA to ensure that the PCR efficiency for all primers is between90% and 110%; (ii) melting curve analysis to ensure single peak of amplifiedproduct with expected melting temperature; and (iii) agarose gel electro-phoresis to ensure a single band of amplified product with expected size.The same quality control criteria applied to the primers of five cellular ref-erence genes, except using serial dilutions of cDNA sample for the standardcurve analysis. For analysis, cells were lysed in TRIzol and RNA was extractedusing Direct-zol RNA kit (Zymo Research). A total of 200 ng of total RNA wasreverse transcribed with qScript cDNA SuperMix (Quanta Biosciences) ina volume of 20 μL and diluted to a volume of 200 μL. The qPCR reactionincluded 0.7 μL of 200-fold diluted cDNA sample from the RT reaction, 400nM of each forward and reverse primer, and SYBR Green master mix ina final volume of 10 μL. The geometric mean of cycle threshold (Ct) values(Table S1) of the five reference genes (PPIA, PGK1, ACTB, HPRT1, and B2M)was used to normalize the viral gene expression at different time pointspostinfection in control or shRNA knockdown cells.

To quantify accumulation of the viral genome by qPCR, cells were washedonce in PBS, and genomic DNA was harvested using the Quick-gDNA kit(Zymo Research) according to the manufacturer’s protocol. Viral genome andcellular genome were quantitated by qPCR using primers recognizing UL123and β-actin, respectively. Standard curves were generated using 10-folddilutions of BFX-actin (76).

Metabolomic Analysis. At 48 h after mock or ADwt infection, medium wasremoved and cellular metabolism was immediately quenched with 80:20methanol/water (vol/vol) solution at −80 °C (45). Extracts were scraped intoa conical tube held on dry ice and centrifuged at 3,200 × g for 5 min. Extractswere dried under nitrogen gas at room temperature, resuspended in water(HPLC grade), and centrifuged at 16,000 × g for 5 min. Metabolites wereidentified and measured by an Orbitrap mass spectrometer (Thermo Scien-tific Exactive) (77), and Metabolomic Analysis and Visualization Engine(MAVEN) software was used for the quantitative analysis of metabolitelevels (66, 78, 79). Metabolite identities were determined by exact mass andretention time match to authenticated standards. Isomeric metabolites mayor may not separate chromatographically. Reported values reflect the sumof the stated metabolite and any nonseparated isomers. Values acquiredfrom experimental conditions were divided by values from the indicatedcontrol conditions to obtain ratios and log2 transformed; a value of zeroindicates no change in metabolite levels.

Statistical Analysis. Values are expressed as mean ± SD, and significance wasdetermined by Student t test. For metabolic data, correlation coefficients wereused to determine the significance of similarities between metabolic profiles.

ACKNOWLEDGMENTS. We thank L. Terry and S. Grady for technical adviceand critical commentary. This work was supported by a Grant AI97382 fromthe National Institutes of Health. J.G.P. was supported by American HeartAssociation Postdoctoral Fellowship 12POST9190001.

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Hwang et al. PNAS | Published online December 15, 2014 | E5715

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