Epstein-Barr Virus-Encoded BILF1 Is a Constitutively Active G Protein-Coupled Receptor

Both beta- and gammaherpesviruses encode G protein-coupled receptors(GPCRs) with unique pharmacological phenotypes and importantbiological functions. An example is ORF74, the ${gamma}$ 2-herpesvirusKaposi's sarcoma-associated herpesvirus (KSHV)-encoded GPCR,which is highly constitutively active and considered the keyoncogene in Kaposi's sarcoma pathogenesis. In contrast, thecurrent annotation of the Epstein-Barr virus (EBV) genome doesnot reveal any GPCR homolog encoded by this human oncogenic ${gamma}$ 1-herpesvirus. However, by employing bioinformatics, we recognizedthat the previously established EBV open reading frame BILF1indeed encodes a GPCR. Additionally, BILF1 is a member of anew family of related GPCRs exclusively encoded by ${gamma}$ 1-herpesviruses.Expression of hemagglutinin-tagged BILF1 in the HEK293 epithelialcell line revealed that BILF1 is expressed as an approximately50-kDa glycosylated protein. Immunocytochemistry and confocalmicroscopy revealed that BILF1 localizes predominantly to theplasma membrane, similar to the localization of KSHV ORF74.Using chimeric G proteins, we found that human and rhesus EBV-encodedBILF1 are highly potent constitutively active receptors, activatingG ${alpha}$ _i. Furthermore, BILF1 is able to inhibit forskolin-triggeredCREB activation via stimulation of endogenous G proteins ina pertussis toxin-sensitive manner, verifying that BILF1 signalsconstitutively through G ${alpha}$ _i. We suggest that EBV may use BILF1to regulate G ${alpha}$ _i-activated pathways during viral lytic replication,thereby affecting disease progression.

INTRODUCTION

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Introduction

Many beta- and gammaherpesviruses have acquired G protein-coupledreceptors (GPCRs), some of which are functional chemokine receptors(30). While all members of the betaherpesvirus family, e.g.,cytomegalovirus (CMV), encode GPCR homologs, it is the generalnotion that ${gamma}$ 1-herpesviruses, e.g., Epstein-Barr virus (EBV),unlike ${gamma}$ 2-herpesviruses, e.g., Kaposi's sarcoma-associated herpesvirus(KSHV), do not encode GPCR homologs (46). Most virus-encodedGPCRs, including UL33, M33, R33, UL78, M78, R78, US27, and US28(6, 7, 10, 18, 41, 42, 60), are dispensable for viral growthin tissue culture. However, several in vivo studies have revealedthat viral GPCRs are highly significant for viral replicationand for virus-induced pathogenesis in the natural hosts (6,7, 18, 42, 49, 50). For example, M33-deficient murine CMV doesnot disseminate to the salivary glands of infected mice (18),R33-deficient rat CMV is less virulent for immunocompromisedrats than wild-type virus, and in parallel to M33 mutant murineCMV, R33 mutant rat CMV does not replicate efficiently in thesalivary glands of infected rats (7). Furthermore, R78-deficientrat CMV is less virulent than wild-type rat CMV (6), and M78deletion mutant murine CMV replicates and/or disseminates toa lower level in the salivary glands, spleen, and liver in bothimmunocompetent and immunocompromised mice (42).

${gamma}$ 2-Herpesvirus-encoded GPCRs also have important functions. Murinegammaherpesvirus ORF74 knockout virus suffers from decreasedefficiency of reactivation from latency both in vitro and invivo compared to wild-type virus (32, 39). This is surprisingbecause ORF74 is regarded as an early lytic gene and is notexpressed in the vast majority of otherwise latently infectedcells (14, 29). This suggests that lytic replication can transformlatently infected cells, presumably through paracrine mechanisms.The activities and biological effects of the KSHV-encoded chemokinereceptor ORF74 are well characterized. ORF74 is highly constitutivelyactive (1, 3, 22, 48) and mediates its signals through severaldifferent G ${alpha}$ proteins and by activating ß ${gamma}$ subunits.As a result, ORF74 triggers several of the major signaling transductionpathways, including the phospholipase C and protein kinase Cpathways (1, 48, 54), the phosphoinositol-3'-kinase-AKT/proteinkinase B pathway (38, 54), and the mitogen-activated proteinkinase pathways JNK, p38 (3), and p44/p42 MAPK (52, 54). Consequently,ORF74 induces various growth factors and angiogenic and proinflammatorycytokines (38, 44, 45, 52, 53). Through activation of vascularendothelial growth factor, ORF74 stimulates the proliferationof transfected cells (1, 3) and induce angiogenesis of humanumbilical vein endothelial cells (3, 57). Injection of ORF74-expressingmouse fibroblasts into the flank of nude mice causes vascularizedtumors (3), and most significantly, transgenic mice expressingORF74 ubiquitously or within hematopoietic or endothelial cellsdevelop Kaposi's sarcoma-like lesions in multiple organs (24,26, 37, 63). Therefore, ORF74 is considered the key viral oncogenein Kaposi's sarcoma pathogenesis.

In this paper we describe that the EBV-encoded open readingframe (ORF) BILF1 encodes a functional GPCR. The EBV genomewas sequenced and annotated 20 years ago (2). The EBV annotationis based on BamHI fragments, and the BILF1 ORF is localizedto the small BamHI fragment I. At least two lytic transcripts,a 1.1-kb and a 1.4-kb transcript, are known to be expressedfrom this region (27). We discovered that BILF1 contained severalhallmarks of GPCRs, including seven hydrophobic transmembranedomains, conserved cysteines in the amino terminus and in theextracellular loops, amino-terminal glycosylation sites, andintracellular phosphorylation sites. We therefore set out totest whether BILF1 was a functional GPCR. We show that BILF1is expressed as a heavily glycosylated membrane protein andthat BILF1 is a highly potent GPCR, constitutively signalingthrough G ${alpha}$ _i. Given the importance of ORF74-mediated activityfor ${gamma}$ 2-herpesvirus replication and for the development of Kaposi'ssarcoma, it is intriguing that the oncogenic ${gamma}$ 1-herpesvirusesalso encode constitutively active GPCRs. Because of this newGPCR similarity between the two human oncogenic gammaherpesviruses,further research into the activities and functions of BILF1is important. Not only will it result in a better understandingof the significance of lytic gene products for gammaherpesvirusreplication and reactivation, it may also give clues to whyboth ${gamma}$ 1- and ${gamma}$ 2-herpesviruses express GPCRs with similar pharmacologicalcharacteristics during lytic replication.

MATERIALS AND METHODS

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Materials and Methods

Bioinformatics. The BLAST program available at http://www.ncbi.nlm.nih.gov/BLAST/was used to identify EBV-encoded sequences with similaritiesto known GPCRs. The program TMHMM was used to predict transmembranehelices, NetNGlyc and NetOGlyc were used to predict N-linkedand O-GalNAc glycosylation sites, respectively, and NetPhoswas used to predict phosphorylation sites. All prediction programsare available at http://www.cbs.dtu.dk/services/.

Cell culture, reagents, and plasmids. Human EBV DNA (strain B95.8) and rhesus monkey EBV DNA werekindly provided by Fred Wang, Harvard University, Cambridge,Mass. The glucose-dependent insulinotropic peptide (GIP) receptor,the ${kappa}$ -opiate receptor, the wild-type G ${alpha}$ _q expression construct,GIP, and dynorphin were kindly provided by Christian Elling,7TM-pharma, Hoersholm, Denmark. The recombinant G proteins G ${alpha}$ _qs5and G ${alpha}$ ${Delta}$ _6qi4myr were kindly provided by Evi Kostenis, 7TM-pharma,Hoersholm, Denmark. G ${alpha}$ _qs5 and G ${alpha}$ ${Delta}$ _6qi4myr have switched receptorspecificity from wild-type G ${alpha}$ _q-interacting receptors to G ${alpha}$ _s- andG ${alpha}$ _i-interacting receptors, respectively; G ${alpha}$ _qs5 has replaced thefive C-terminal amino acids of wild-type G ${alpha}$ _q with the five correspondingC-terminal amino acids from G ${alpha}$ _s, and G ${alpha}$ ${Delta}$ _6qi4myr lacks the firstsix N-terminal amino acids, replacing the four C-terminal aminoacids of wild-type G ${alpha}$ _q with the corresponding four C-terminalamino acids from G ${alpha}$ _i, and including an N-terminal myristoylationsite (15, 31, 51).

The T-REx-293 cell line (Invitrogen R710-07) was grown in Dulbecco'smodified Eagle's medium (Invitrogen 31966-021) plus 10% fetalbovine serum and penicillin-streptomycin (Invitrogen 15140-022)with the addition of 15 µg of blasticidin S (InvitrogenR210-01) per ml and 100 µg of zeocin (Invitrogen R250-01)per ml or 150 µg of hygromycin B (Invitrogen 10687-010)per ml at 37°C and 5% CO₂. COS-7 cells were grown in Dulbecco'smodified Eagle's medium (Invitrogen 21885-025), 10% fetal bovineserum, and penicillin-streptomycin at 37°C and 10% CO₂.

Receptor cloning. Cloning of human and rhesus EBV BILF1 and receptor hemagglutinintagging was done with standard PCR techniques with PFU polymerase(Stratagene) with, for human EBV BILF1, forward primer 5'-TAGAAGCTTATGTACCCATACGACGTACCAGACTACGCACTCTCCACCATGGCCCCCand reverse primer 5'-TAGCTCGAGTCAGGTGGACTGGCTAGGC and, forrhesus EBV BILF1, forward primer 5'-TAGAAGCTTATGTACCCATACGACGTACCAGACTACGCACTCTCCACCCTGGCCCCand reverse primer 5'-TAGCTCGAGTCAGGTGGACTGGGTGGAC). Boldfacetype indicates the start codon for methionine; italic type indicatesthe part of the primer that overlaps the template gene. Humanand rhesus EBV BILF1 were inserted into pcDNA5/FRT/TO (InvitrogenV6520-20) by cohesive end ligation. The resulting human andrhesus EBV BILF1 constructs (BILF1-pcDNA5/FRT/TO) are hereaftercalled HuBILF1 and RhBILF1, respectively. The constructs alsocontain the hygromycin resistance gene with an flp recombinationtarget (FRT) site embedded in the 5' coding region. The hygromycinresistance gene lacks a promoter and the ATG initiation codon

Generation of stable, BILF1-inducible cell lines. Stable, tetracycline-inducible BILF1 cell lines were generatedwith the Flp-In T-Rex Core kit (Invitrogen K6500-01) by flprecombinase-mediated integration. The 293 T-REx cell line containsone integrated FRT site originally encoded by the pFRT/lacZeovector (Invitrogen V6015-20) and expresses the tetracyclinerepressor originally encoded by the pcDNA6/TR vector (InvitrogenV1025-01). The FRT site is maintained by selection for zeocinresistance, and the tetracycline repressor is maintained byselection for blasticidin resistance. The integrated FRT siteis contained just downstream of the ATG initiation codon ofthe lacZ-zeo fusion gene. Briefly, T-REx-293 cells were transfectedwith HuBILF1 or RhBILF1 and pOG44 (Invitrogen V6005-20) fortransient expression of the Flp recombinase with the Fugene-6transfection reagent (Roche 1814433) according to the manufacturer'sprotocol. Upon cotransfection, the Flp recombinase mediateshomologous recombination between the FRT site, so that BILF1is inserted into the genome at the already integrated FRT site.This insertion brings the simian virus 40 promoter and the ATGinitiation codon (from the lacZ-zeo fusion gene) into proximityand in frame with the ATG minus hygromycin resistance gene,and at the same time inactivates the lacZ-zeo fusion gene. StableBILF1 clones were selected for blasticidin and hygromycin resistanceand controlled for zeocin sensitivity and screened for the lackof ß-galactosidase activity. The resulting HuBILF1and RhBILF1 cell lines therefore contain the BILF1 ORF underthe control of a tetracycline-regulated hybrid CMV/tetO₂ promoter.The CMV/tetO₂ promoter is inactive in cells expressing the tetracyclinerepressor (T-REx-293 cell line), where it is activated by theaddition of tetracycline. It should be noted that the CMV/tetO₂promoter is fully active in cells not expressing the tetracyclinerepressor.

Immunoblotting and receptor deglycosylation. For receptor studies, cells were stimulated with tetracycline(Invitrogen Q100-19) to induce receptor expression. For Westernblotting, cells were lysed in radioimmunoprecipitation assay(RIPA) lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecylsulfate with the addition of protease inhibitors [phenylmethylsulfonylfluoride, aprotinin, leupeptin, and pepstatin] and phosphataseinhibitors [activated Na₃VO₄ and NaF]). For receptor glycosylationstudies, cells were lysed in 0.1 M Tris-HCl-10 mM EDTA-1% TritonX-114-1 mM phenylmethylsulfonyl fluoride. Triton X-114 is solublein aqueous solutions at 0°C but not at 37°C. This propertymakes is suitable for separating membrane fractions (and theembedded receptors) from water-soluble proteins. Briefly, cellswere lysed on ice for 15 min and centrifuged for 30 min at 5,500rpm at 4°C. The supernatant was incubated at 37°C untilunclear and spun for 10 min at 4,000 rpm at room temperatureto separate the detergent phase from the aqueous phase (upperphase). The detergent phase was supplemented with 0.1 M Tris-HCl(pH 8.1) to starting volume and incubated on ice until clarified,and the phase separation was repeated. The detergent phase wasagain supplemented with 0.1 M Tris-HCl (pH 8.1) to startingvolume and clarified with 2.5 µl of 10% CHAPS. The supernatantdetergent phase was separated from the aqueous phase by centrifugationfor 15 min at 4,000 rpm at 4°C. Receptor deglycosylationwas done with glycopeptidase F (Sigma G5166) according to themanufacturer's protocol, and proteins were analyzed by Westernblotting with antihemagglutinin antibody (Biosite HA.11). Fordetection of the internal control, glyceraldehyde-3-phosphatedehydrogenase, blots were stripped in Restore Western blot strippingbuffer (Pierce 21059) for 20 min at room temperature and analyzedwith mouse anti-rabbit glyceraldehyde-3-phosphate dehydrogenaseantibody (Research Diagnostics RDI-TRK5G4-6C5).

Immunocytochemistry and confocal microscopy. For immune staining, HuBILF1 and RhBILF1 cell lines were grownon CC2-treated Lab-Tek chamber glass slides (Nalge Nunc International154917). One day after seeding into the chamber glass slides,the cells were transfected with CXCR4-GFP²⁰, US28-GFP²⁰ or ORF74-GFP³⁵with Fugene 6 transfection reagent (Roche 1 814 443) accordingto the manufacturer's recommendations. The following day, cellswere stimulated with tetracycline (0.1 µg/ml); 24 h aftertetracycline stimulation, cells were fixed in 3.7% formaldehyde,washed in phosphate-buffered saline, and permeabilized in 0.2%Triton X-100 for 20 min on ice. Cells were blocked in phosphate-bufferedsaline containing 1% bovine serum albumin and 5% goat serumfor 30 min at room temperature and incubated with antihemagglutininantibody (Biosite HA.11) for 1 h at room temperature. Cellswere washed and incubated with Alexa Fluor 594-conjugated goatanti-mouse immunoglobulin antibody (Molecular Probes A-11005)for 1 h at room temperature. Cells were mounted with Fluoromount-G(Southern Biotechnology Associates 0100-01). Confocal fluorescencemicroscopy was done on an inverse Zeiss Axiovert 200 fluorescencemicroscope equipped with a Coolsnap charge-coupled device camera.Digital images were transferred to Adobe Photoshop and usedwithout further processing.

Inositol phosphate production. For inositol phosphate turnover, COS-7 cells were transfectedwith the calcium phosphate precipitation method (48). Briefly,20 µl of 2 M CaCl₂, DNA, and Tris-EDTA buffer to a totalvolume of 160 µl were mixed and added dropwise to 160µl of 2x HEPES-buffered saline buffer. After 45 min ofincubation, the mixture was added to the cells and incubatedwith the addition of 100 µM chloroquine for 5 h. One dayafter transfection, the cells were transferred to six-well plates(5 x 10⁵ cells/well) and incubated for 24 h with 4 µCiof myo-[³H]inositol (Amersham TRK911) in 0.8 ml of completemedium/well. Cells were washed twice in immunoprecipitationbuffer (20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl,5 mM KCl, 1 mM MgSO₄, 1 mM CaCl₂, 10 mM glucose, and 0.05% bovineserum albumin) and incubated in 1.0 ml of immunoprecipitationbuffer supplemented with 10 mM LiCl at 37°C for 90 min.GIP and dynorphin were added to a final concentration of 10^–7M after 15 min of incubation. After incubation, the buffer wasremoved, and accumulated inositol phosphates were extractedfor 30 min on ice in 1.0 ml of 10 mM formic acid. The generated[³H]inositol phosphates were purified on Dowex 1X8 anion-exchangeresin, and radioactivity was counted in a scintillation counter(48). Radioactivity values are given as counts per minute.

CREB reporter assay. For luciferase reporter assays, COS-7 cells (35,000 cells/well)were seeded in 96-well plates and transfected with a reporter-cDNAcocktail consisting of 6 ng of pFA2-CREB and 50 ng of pFR-Lucreporter plasmids with the PathDetect cyclic AMP response elementbinding protein (CREB) system (Stratagene) and various amountsof receptor and G protein DNA with Lipofectamine 2000 (Invitrogen)according to the manufacturer's protocol. Following transfections,cells were treated with 10 µM forskolin or 10 µMforskolin plus 0.1 µg of pertussis toxin per ml in anassay volume of 100 µl for 24 h. The assay was terminatedby washing the cells twice with phosphate-buffered saline andadding 100 µl of phosphate-buffered saline supplementedwith 1 mM MgCl₂ and CaCl₂ and 100 µl of luciferase assayreagent (LucLite; Packard). Luminescence was measured in a TopCounter(Top Count NXT; Packard) for 5 s. Luminescence values are givenas relative light units.

RESULTS

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Results

EBV encodes a GPCR homolog. Initially, BLAST analysis revealed that the equine herpesvirus2-encoded GPCR homolog, ORF E6, contained limited sequence homology(17% amino acid identity, PAM250 similarity matrix) to EBV ORFBILF1. Transmembrane helix analysis clearly demonstrated thatBILF1 contains seven hydrophobic helices (Fig. 1), a hallmarkof all GPCRs. In addition, BILF1 has several additional characteristicsof GPCRs. These include conserved cysteines in the amino-terminalend and in the extracellular loops (Fig. 1), which are knownto form structurally and functionally important disulfide bondsin GPCRs (19). Furthermore, BILF1 is predicted to contain sevenN-terminal glycosylation sites, which are important for GPCR-ligandinteractions, receptor expression, and cellular localization(33), as well as four intracellular phosphorylation sites (Fig.1), which are important for GPCR-mediated signaling, receptorregulation, and intracellular targeting (36). Interestingly,the DRY (aspartic acid, arginine, tyrosine) motif at the intracellularend of TM-III, which is conserved in most rhodopsin-like GPCRs,is replaced with an EKT (glutamic acid, lysine, threonine) motifin BILF1. However, this conservative amino acid substitution(same order of acidic, basic, and polar side chains) can beconsidered an alternative DRY motif (Fig. 1 and 2). It shouldbe noted that alternative DRY box motifs are present in numerousviral GPCRs.

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FIG. 1. Serpentine diagram of the human EBV BILF1 receptor, showing the seven transmembrane helices. Differences between human EBV and rhesus EBV are indicated in black on grey, and identical amino acids are indicated in black on white. Computer-predicted glycosylation sites are indicated with g, and predicted phosphorylation sites are indicated with p. The alternative DRY box, EKT, is marked with a rectangle.

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FIG. 2. Multiple sequence alignment of BILF1-related sequences identified with BLAST. The alignment was done with ClustalW version 1.8, and shading was done with Boxshade version 3.21, available at http://www.ch.embnet.org/software/BOX_form.html. Abbreviations used in this figure: PLHV, porcine lymphotropic herpesvirus; CHV3, callitrichine herpesvirus 3 (also known as marmoset EBV); AHV, alcelaphine herpesvirus; EHV, equine herpesvirus. The rectangle indicates the DRY box. Positions sharing 50% amino acid identity or more are indicated in white on black. Positions sharing 50% amino acid homology or more are indicated in black on gray. Positions with less than 50% amino acid identity or homology are indicated in black on white. The consensus line asterisk indicates 100% conserved positions, and dots indicate positions with 50% or better homology or identity.

BILF1 belongs to a novel receptor subfamily. Further BLAST analysis revealed that BILF1 is encoded not onlyby human EBV but also by other ${gamma}$ 1-herpesviruses. Together, thisgroup of BILF1-related sequences constitute a new family ofrelated GPCRs, exclusively encoded by and a trait for ${gamma}$ 1-herpesviruses(Fig. 2). Indeed, BILF1 is one of just six genes present onlyin ${gamma}$ 1-herpesviruses (47). This genomic evidence suggests thatBILF1 plays an important role in the life cycle of ${gamma}$ 1-herpesviruses.Multiple sequence alignment of eight BILF1-related sequenceswith a wide range of viral GPCRs belonging to the US28, UL33,UL78, UL51, and ORF74 subfamilies revealed that all BILF1 sequencesgroup together and constitute a separate viral GPCR subfamily(Fig. 3)

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FIG. 3. Dendrogram of herpesvirus-encoded G protein-coupled receptors based on their amino acid identities. Receptor alignment was generated in Clustal X (1.81), and the phylogenetic tree was visualized with TreeView. The length of each branch reflects amino acid discrepancies. Each entry consists of the abbreviated virus name followed by the name of the reading frame encoding the receptor. The BILF1 receptor family is highlighted in bold and consists of porcine lymphotrophic herpesvirus (PLHV) types 1, 2 and 3 ORF A5, alcelaphine herpesvirus 1 (AHV) ORF E5, equine herpesvirus 2 (EHV2) ORF E6, rhesus Epstein-Barr virus (RhEBV) ORF BILF1, human Epstein-Barr virus (EBV) ORF BILF1, and callitrichine herpesvirus 3 (CHV3) ORF 6.

Cloning and generation of cell lines. We cloned human EBV and rhesus EBV BILF1 and inserted the receptorreading frames into pCDNA5/FRT/TO, generating HuBILF1 and RhBILF1,respectively. The BILF1 sequences were confirmed by sequenceanalysis and corresponded to nucleotides 152161 to 153099 inthe human herpesvirus 4 complete genome (GenBank accession no.NC_001345) and to nucleotides 147746 to 148684 in the rhesusEBV (cercopithicine herpesvirus 15) complete genome (GenBankaccession no. AY037858). We generated several clonal cell linesof both HuBILF1 and RhBILF1. For our expression studies, weselected clones with the lowest BILF1 background expressionthat could be induced to express high levels of BILF1 aftertetracycline induction. Tetracycline dose experiments showedthat 0.1 µg of tetracycline/ml of medium was sufficientto induce maximal BILF1 expression 24 h poststimulation (datanot shown).

BILF1 is heavily glycosylated. To study receptor glycosylation, membrane samples from tetracycline-inducedHuBILF1-expressing cell lines were either untreated or treatedwith glycopeptidase F to remove N-linked glycosylation sitesprior to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) separation and Western blotting. Staining with antihemagglutininantibody showed that BILF1 is expressed as a heterogeneous approximately50 kDa heavily glycosylated protein since glycopeptidase F treatmentreduced the apparent mass to approximately 33 kDa, which isclose to the calculated mass of 34.5 kDa (Fig. 4). These datasupport the computer-based prediction of N-terminal glycosylationsites in BILF1.

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FIG. 4. Western blot of tetracycline-induced HuBILF1 cell lines. Gel 1: Total radioimmunoprecipitation-extracted protein were boiled and reduced with 100 mM dithiothreitol prior to SDS-PAGE separation and Western blotting. A) Tetracycline-stimulated HuBILF1 cells, B) unstimulated HuBILF1 cells, C) tetracycline-stimulated parental HEK293 Flp In T-Rex cells, and D) unstimulated parental HEK293 Flp In T-Rex cells. The lower image shows the same plot after antibody stripping and restaining with an anti-glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) antibody for loading control. Gel 2: Detergent phase samples were either (A) untreated, (B) boiled, (C) reduced with 100 mM dithiothreitol (DTT), or boiled and reduced (D) without or (E) with glycopeptidase F treatment prior to SDS-PAGE separation and Western blotting. Nondeglycosylated BILF1 is expressed as an approximately 50-kDa protein. Glycopeptidase F treatment reduced the apparent mass to approximately 33 kDa.

BILF1 is localized to the plasma membrane. Tetracycline-induced HuBILF1 and RhBILF1 cell lines were stainedwith an antihemagglutinin antibody, and receptor localizationwas studied by confocal microscopy. As shown in Fig. 5, BILF1localizes mainly to the plasma membrane. This expression patternis concurrent with the expression pattern of most GPCRs, butis in contrast to the predominantly intracellular localizationof many viral GPCRs (30). BILF1 colocalizes with the human CXCchemokine receptor CXCR4 and with the KSHV ORF74 to the cellplasma membrane, whereas BILF1 localizes differently than US28,which has a distinct intracellular accumulation. These datasupport our prediction that BILF1 is indeed a membrane proteinand implies that our cell lines express properly folded hemagglutinin-taggedBILF1.

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FIG. 5. Cellular localization of human and rhesus EBV BILF1. Representative images of HuBILF1-expressing 293 cells cotransfected with either A) CXCR4-GFP, B) ORF74-GFP, or C) US28-GFP. Cells were fixed, permeabilized, and stained with an antihemagglutinin antibody. The images were taken with a 60X oil immersion objective. The images show that BILF1 has a distinct receptor membrane association similar to that of CXCR4 and ORF74 and different from the predominantly intracellular localization of US28.

BILF1 signals constitutively through G ${alpha}$ _i. To study whether human EBV BILF1 is a functional GPCR, we testedthe activation of a variety of G ${alpha}$ proteins. To facilitate thesestudies of constitutive, i.e., ligand-independent, BILF1-mediatedsignaling, we used chimeric G ${alpha}$ _q proteins comprising the threemajor types of G ${alpha}$ subunits; G ${alpha}$ _q, G ${alpha}$ _s and G ${alpha}$ _i. Both the wild-typeand chimeric G ${alpha}$ _q proteins G ${alpha}$ _qs5 and G ${alpha}$ ${Delta}$ _6qi4myr signal via activationof phospholipase C, leading to accumulation of [³H]inositolphosphates. COS-7 cells were transfected with an increasingamount of HuBILF1 DNA together with a constant quantity of differentG ${alpha}$ DNA or empty expression vector DNA. The Western blot in Fig.6 show that an increase in BILF1 DNA corresponds to an increasein BILF1 protein expression. Figure 6A shows that BILF1 didnot activate phospholipase C through endogenous G ${alpha}$ _q. As a positivecontrol for endogenous G ${alpha}$ _q function, we transfected COS-7 cellswith KSHV ORF74, which constitutively activates G ${alpha}$ _q. As shownin Fig. 6B, transfected COS-7 cells were fully capable of activatingphospholipase C through endogenous G ${alpha}$ _q. Cotransfection with G ${alpha}$ _qs5revealed that BILF1 does not signal via activation of G protein ${alpha}$ subunits of the s class (Fig. 6A). As a positive control forG ${alpha}$ _s function, we cotransfected COS-7 cells with G ${alpha}$ _qs5 and theGIP receptor (which activates G ${alpha}$ _s upon ligand engagement) andstimulated transfected cells with GIP. As shown in Fig. 6C,G ${alpha}$ _qs5 was fully capable of activating phospholipase C after GIP-receptoractivation. Finally, we investigated whether BILF1 could signalthrough G protein ${alpha}$ subunits of the i class. Cotransfection withG ${alpha}$ ${Delta}$ _6qi4myr revealed that BILF1 indeed signals through G protein ${alpha}$ subunits of the i class. Figure 6A shows that increasing dosesof BILF1 increase phospholipase C activity only when cotransfectedwith chimeric G protein with specificity towards G ${alpha}$ _i-activatingreceptors. As an additional positive control for G ${alpha}$ _i function,we cotransfected COS-7 cells with the ${kappa}$ -opiate receptor and G ${alpha}$ ${Delta}$ _6qi4myrand stimulated transfected cells with dynorphin. As seen inFig. 6C, G ${alpha}$ ${Delta}$ _6qi4myr was also fully capable of activating phospholipaseC after ${kappa}$ -opiate receptor activation. Importantly, none of thechimeric and endogenous G proteins were active without concurrentreceptor expression (Fig. 6A, B, and C). To further test thespecificity of the system and to ensure that our results werenot merely a result of G protein overexpression, we cotransfectedBILF1 with wild-type G ${alpha}$ _q. Transfection with G ${alpha}$ _q did not resultin an increased basic level of phospholipase C activity, nordid it cause any measurable BILF1 activity (Fig. 6A and B),whereas overexpression of G ${alpha}$ _q slightly increased ORF74-mediatedsignaling (Fig. 6B). Overall, these data show that BILF1 isconstitutively active and mediates its signal through G protein ${alpha}$ subunits of the i class.

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FIG. 6. HuBILF1 induction of inositol phosphate accumulation. COS-7 cells were transfected with an increasing amount of HuBILF1 DNA together with 5 µg of different chimeric G ${alpha}$ protein DNA or empty expression vector DNA. The recombinant G proteins G ${alpha}$ _qs5 and G ${alpha}$ ${Delta}$ _6qi4myr have switched receptor specificity from wild-type G ${alpha}$ _q interacting with receptors to G ${alpha}$ _s- and G ${alpha}$ _i-interacting receptors, respectively, but all signal via activation of phospholipase C, leading to accumulation of inositol phosphates (IP3). Panel A shows the activity as a percentage of maximum activity obtained. HuBILF1 plus control DNA (open squares), HuBILF1 plus wild-type G ${alpha}$ _q DNA (solid diamonds), HuBILF1 plus G ${alpha}$ _qs5 DNA (solid triangles), and HuBILF1 plus G ${alpha}$ ${Delta}$ _6qi4myr DNA (solid squares). Data are means and standard deviations of four independent experiments, each experiment carried out in duplicate. The Western blot shows the expression levels of BILF1 in transfected COS-7 cells, with the amount of DNA given above each lane. The amount of DNA in the Western blot corresponds to the amount of DNA used in the [³H]inositol phosphate assay shown above. The lower Western blot image shows the same plot after antibody stripping and restaining with an anti-glyceraldehhyde-3-phosphate dehydrogenase (GAPDH) antibody as a loading control. To illustrate both the magnitude and specificity of the BILF1-mediated response, panels B and C show the actual counts from one representative experiment out of four. Cells were transfected with 10 µg of receptor DNA and 5 µg of G protein DNA and stimulated with ligands as indicated below the graphs. Bars illustrate means and error bars illustrate standard deviations of one experiment carried out in duplicate. BILF1 is HuBILF1, ORF74 is the KSHV-encoded GPCR, qi4 is G ${alpha}$ ${Delta}$ _6qi4myr, qwt is wild-type G ${alpha}$ _q, qs5 is G ${alpha}$ _qs5, GIP (receptor) is the glucose-dependent insulinotropic peptide receptor, ${kappa}$ OP (receptor) is the ${kappa}$ -opiate receptor, GIP (ligand) is the glucose-dependent insulinotropic peptide, and Dyn (ligand) is dynorphin. GIP and dynorphin were added to a final concentration of 10^–7 M.

We further tested whether rhesus EBV-encoded BILF1 was alsoconstitutively active and whether rhesus EBV BILF1 activatedthe same G protein as the human EBV BILF1. Cotransfection ofRhBILF1 with the chimeric G proteins described above revealedthat rhesus EBV BILF1 is also constitutively active and, likethe human EBV homolog, mediates its signal through activationof G ${alpha}$ _i. Figure 7 shows a representative example of four independentexperiments. Besides revealing homologous G ${alpha}$ subunit activation,the RhBILF1 activity level was very similar to the activityof HuBILF1 (compare Fig. 6B and 7). Thus, both human and rhesusEBV BILF1 signal constitutively via G ${alpha}$ _i with similar efficacies.

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FIG. 7. RhBILF1 induction of inositol phosphate accumulation. COS-7 cells were transfected with 6 µg of BILF1 DNA together with 5 µg of different chimeric G ${alpha}$ protein DNA or empty expression vector DNA as indicated below the graph. The figure shows actual counts from one representative experiment out of four. Bars illustrate means and error bars illustrate standard deviations of one experiment carried out in duplicate.

As an additional readout for BILF1-driven G ${alpha}$ _i activity, we measuredthe generation of CREB-stimulated luciferase reporter activity.COS-7 cells were transfected with an increasing amount of HuBILF1DNA together with a constant quantity of G ${alpha}$ ${Delta}$ _6qi4myr DNA or emptyexpression vector and a reporter-cDNA cocktail consisting of6 ng of pFA2-CREB and 50 ng of pFR-Luc reporter plasmids. Figure8A shows that BILF1 activates CREB through G ${alpha}$ ${Delta}$ _6qi4myr in a dose-dependentmanner. Furthermore, it is evident that BILF1 does not activateCREB without cotransfection with G ${alpha}$ ${Delta}$ _6qi4myr. This result thereforeconfirms the inositol phosphate data, validating that BILF1signals constitutively through G ${alpha}$ _i.

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FIG. 8. CREB-mediated transcription regulation. (A) COS-7 cells were seeded in 96-well plates and cotransfected with a reporter-cDNA cocktail consisting of 6 ng of pFA2-CREB and 50 ng of pFR-Luc reporter plasmids and increasing amounts of HuBILF1 DNA with (solid squares) or without (open squares) 30 ng of G ${alpha}$ ${Delta}$ _6qi4myr DNA. Data points illustrate means and error bars illustrate standard deviations of one experiment out of four, each carried out in triplicate. Luciferase activity is presented as relative light units (RLU). (B) COS-7 cells were seeded in 96-well plates and cotransfected with a reporter-cDNA cocktail consisting of 6 ng of pFA2-CREB and 50 ng of pFR-Luc reporter plasmids and increasing amounts of receptor DNA and incubated in the presence of 10 µM forskolin to stimulate the adenylyl cyclase, with (white bars) or without (solid bars) the addition of 0.1 µg of pertussis toxin per ml to inhibit endogenous G ${alpha}$ _i. Data are presented as a percentage of the CREB activity generated by 10 µM forskolin alone above the values obtained for unstimulated HuBILF1-transfected cells.

BILF1 activates endogenous i-class G proteins. To study whether BILF1 could activate endogenous G ${alpha}$ _i, we testedthe ability of BILF1 to inhibit forskolin-stimulated CREB activity.COS-7 cells were transfected with increasing amounts of HuBILF1DNA together with a reporter-cDNA cocktail consisting of 6 ngof pFA2-CREB and 50 ng of pFR-Luc reporter plasmids. After transfection,cells were stimulated with 10 µM forskolin with or withoutthe addition of PTx. Figure 8B show that BILF1 almost completelyinhibited forskolin-induced CREB activity in a dose-dependentmanner. Furthermore, BILF1-mediated signaling can be inhibitedby addition of PTx, a potent inhibitor of G ${alpha}$ _i. The addition ofPTx restored CREB activity to approximately 60% of maximum forskolin-stimulatedCREB activity. These data confirms that BILF1 constitutivelyactivates endogenous G proteins of the i class.

DISCUSSION

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Discussion

Several large DNA viruses encode viral GPCRs with unusual pharmacologicaland cellular properties and significant biological functions.We have identified a new subfamily of viral GPCRs, the BILF1receptors, encoded by EBV and other ${gamma}$ 1-herpesviruses. Our studiesof human EBV BILF1 and the closely related homolog from rhesusEBV show that these receptors are functional GPCRs, constitutivelysignaling through G ${alpha}$ _i. Additionally we show that BILF1 is heavilyglycosylated and is localized to the plasma membrane in stablyexpressing epithelial cell lines. It is intriguing that EBV,a major human oncogenic herpesvirus, encodes a constitutivelyactive GPCR, considering the significance of the KSHV-encodedGPCR, ORF74, for the development of Kaposi's sarcoma. However,even though the significance of ORF74 for Kaposi's sarcoma pathogenesisis well established, many questions regarding its significancefor viral replication still remain unanswered.

One interesting function of ORF74 may be to increase the efficiencyof KSHV reactivation (39). This observation is intriguing becauseORF74 is regarded as an early lytic gene and is not expressedin the vast majority of otherwise latently infected cells (13),which suggests that some level of lytic replication can activatelatently infected cells, presumably through paracrine mechanisms.Intriguingly, it has been proposed that dysregulation of theKSHV gene program caused by human immunodeficiency virus type1 Tat, inflammation, or aborted lytic replication may indeedlead to nonlytic expression of ORF74 (55). Like ORF74, BILF1is a lytic gene. BILF1 is expressed from 8 h postinduction oflytic replication and throughout infection as an approximately1.1-kb transcript. Furthermore, BILF1 expression is dependenton viral DNA replication, since expression in the Akata B-cellline is inhibited by phosphonoacetic acid (T. Kledal, unpublisheddata).

Primary EBV infection is the most common cause of infectiousmononucleosis, and by adulthood, nearly all humans are asymptomaticcarriers of EBV. Besides causing mononucleosis, in a small percentageof individuals EBV infection is associated with endemic Burkitt'slymphoma, nasopharyngeal carcinoma, Hodgkin's disease, gastriccarcinoma, leiomyosarcoma, and AIDS- and transplant-associatedB-cell lymphomas (25, 46). The viral and host factors implicatedin the development of EBV-associated malignancies are poorlyunderstood. The degree of immunosuppression is one risk factorfor some malignancies (16, 28), but immune suppression aloneis not sufficient for the development of other types of malignancies.Indeed both Burkitt's lymphoma and Hodgkin's lymphoma occurin patients without immunosuppression (46). Additionally, chromosomalabnormalities and somatic hypermutation of cellular oncogeneshave been hypothesized to act in concert with EBV infectionto cause malignancies (5, 43).

Like those associated with KSHV, EBV-associated malignanciesare characterized by a mainly latently infected cell population,and several latently expressed genes have been shown to causecell transformation in vitro. Nevertheless, in a populationof latently EBV-infected cells, a small fraction of cells arespontaneously permissive for lytic replication (46). It is thereforenot impossible that BILF1 could contribute both to control thelevel of reactivation and be involved in EBV tumorigenesis byacting on uninfected or latently infected cells through paracrinemechanisms.

Many viral GPCR (e.g., UL33, US27, US28, and M78) are characterizedby an unusual cellular localization patterns (20, 21, 30). Detailedanalysis of US28 showed that it underwent constitutive endocytosisand recycling to the plasma membrane (20, 21). It has been suggestedthat the constitutive endocytosis and recycling of US28 couldbe a mechanism for sequestering host CC chemokines, providinga sink for clearing proinflammatory CC chemokines from the tissuesurrounding the CMV-infected cell (10), thereby antagonizingthe recruitment of cells involved in the immune response againstCMV. Furthermore, it has been speculated that the intracellularlocalization of many viral GPCRs could facilitate incorporationof the viral GPCRs into the maturing virion envelopes. Indeed,UL33, M78, and US27 have all been identified on viral particles(21, 34, 42). The localization of BILF1 to the plasma membranedistinguishes BILF1 from the UL33, US27, US28, and M78 viralGPCR subfamilies. However, BILF1 localizes similarly to ORF74(52). This parallel could indicate that BILF1 and ORF74 havesimilar functions during gammaherpesvirus replication.

Several receptors from the US28, UL33, and ORF74 receptor subgroupshave been characterized as being constitutively active, a traitof viral GPCRs. As described in the introduction, the KSHV-encodedoncogene ORF74 is highly constitutively active and respondsto ligand engagement. Additionally, UL33, M33, and R33 all constitutivelystimulate phospholipase C, whereas only the rodent CMV receptorsM33 and R33 activate NF- ${kappa}$ B-driven transcription (23, 62). UL33and M33 activate CREB through activation of G ${alpha}$ _s and the mitogen-activatedprotein kinase p38 (62), whereas R33 inhibits forskolin-stimulatedCREB through G ${alpha}$ _i (23). US28 also activates several differentsignal transduction pathways, both constitutively and ligandmediated (8, 12, 58, 59, 61, 62).

Even though a lot is known about viral GPCR pharmacology ingeneral and about what signaling pathways that are activated,little is known about the significance of the constitutive activityfor viral replication. Besides regulating downstream second-messengersystems, controling viral and host gene transcription, and directingcell morphology and motility, the constitutive activity of viralGPCRs may directly regulate the signaling mediated by otherGPCRs (4). If this is indeed the case, BILF1 could functionas a regulatory switch for other GPCRs expressed in EBV-infectedcells. EBV also controls the transcriptional regulation of hostGPCRs. Originally EBV was shown to induce the expression oftwo orphan GPCRs, EBV-induced (EBI) 1 and 2 (9). EBI-1 is nowknown as chemokine receptor CCR7, whereas EBI-2 is still anorphan. In addition, it has been shown that EBV-immortalizedB cells have altered chemokine receptor expression pattern,which could be responsible for the distorted migration of infectedB cells to germinal centers (40). Therefore, EBV seems to controlthe GPCR settings on many levels, emphasizing the importanceof both endogenous GPCRs and viral GPCR for viral replication,dissemination, and immune evasion. It should be noted that BILF1itself does not display any particular similarities to knowchemokine receptors.

Since many receptors activate overlapping second-messenger systems,it is difficult to predict which viral GPCR-activated pathwaysare responsible for cellular transformation. It was recentlyshown that the Akt signaling pathway plays a central role inORF74-mediated oncogenesis (56). Furthermore, it has been shownthat ORF74 constitutively activates Akt via G ${alpha}$ _i and phospholipaseC (11, 17, 54). It is therefore likely that G ${alpha}$ _i activity is directlyinvolved in KSHV-mediated cell transformation, though it isunlikely to be the only signaling pathway involved. Other nontransformingviral GPCRs (e.g., R33 and US28) also activate G ${alpha}$ _i, and ORF74activates several additional pathways. Indeed, the fact thatORF74 stimulates a broad range of signaling pathways has beentaken into account for the transforming properties of this receptor,and it has been suggested that the transforming effects of ORF74may be mediated cooperatively by G ${alpha}$ _q and G ${alpha}$ _i signaling (11, 54).

In this paper we show that the EBV-encoded GPCR BILF1 constitutivelyactivates G ${alpha}$ _i. Given the many similarities between BILF1 andORF74, it is attractive to speculate that BILF1 may play a rolein EBV-associated cell transformation. Additionally, the selectiveand highly constitutive signal triggered by BILF1 suggests thatBILF1 plays a significant role during the life cycle of EBVand possibly during the life cycle of all ${gamma}$ 1-herpesviruses.

ACKNOWLEDGMENTS

This project is supported by Danish Cancer Society grant DP-03-027and by Danish Medical Research council grant 22-03-0181 to T.Kledal. The Danish Cancer Society and the Novo-Nordisk Foundationsupported M. M. Rosenkilde. We also thank Jens Ole Nielsen,Hvidovre Hospital, for generous financial support to the project.

We thank Christian E. Elling and Evi Kostenis, 7TM-pharma, forproviding essential reagents and for helpful discussions duringthis project. We thank Lisbeth Elbak and Inger Smith Simonsenfor excellent technical assistance and Ulrik Gether lab membersfor technical help with confocal microscopy.

FOOTNOTES

* Corresponding author. Mailing address: Clinical Research Unit #136, H:S Hvidovre Hospital, Kettegaards Allé 30-31, 2650 Hvidovre, Denmark. Phone: 45 36322403. Fax: 45 36323797. E-mail: kledal{at}biobase.dk.

REFERENCES

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