Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Production of Infectious Virus

The splicing machinery which positions a protein export complexnear the exon-exon junction mediates nuclear export of mRNAsgenerated from intron-containing genes. Many Epstein-Barr virus(EBV) early and late genes are intronless, and an alternativepathway, independent of splicing, must export the correspondingmRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulationof intronless mRNA, it is tempting to speculate that EB2 isa viral adapter involved in the export of intronless viral mRNA.If this is true, then the EB2 protein is essential for the productionof EBV infectious virions. To test this hypothesis, we generatedan EBV mutant in which the BMLF1 gene, encoding the EB2 protein,has been deleted (EBV_BMLF1-KO). Our studies show that EB2 isnecessary for the production of infectious EBV and that itsfunction cannot be transcomplemented by a cellular factor. Inthe EBV_BMLF1-KO 293 cells, oriLyt-dependent DNA replicationwas greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmicaccumulation of a subset of EBV early mRNAs coding for essentialproteins implicated in EBV DNA replication during the productivecycle. Two herpesvirus homologs of the EB2 protein, the herpessimplex virus type 1 protein ICP27 and, the human cytomegalovirusprotein UL69, only partly rescued the phenotype of the EBV_BMLF1-KOmutant, indicating that some EB2 functions in virus productioncannot be transcomplemented by ICP27 and UL69.

INTRODUCTION

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Introduction

Epstein-Barr virus (EBV) is a human gammaherpesvirus associatedwith several malignancies, such as Burkitt's lymphoma, nasopharyngealcarcinoma, Hodgkin's disease, gastric carcinoma, breast carcinoma,and B- and T-cell lymphomas. EBV also induces the activationand proliferation of quiescent B lymphocytes in vitro. In EBV-associatedtumors in vivo as well as in EBV-infected B cells proliferatingex vivo, the transcription of the EBV genome is restricted toa few genes defining a latent state of the virus (for references,see references 23 and 36).

The mechanisms leading to the switch from latency to the productivecycle are now partially deciphered for in vitro-immortalizedB cells (15, 47). It is accepted that the expression of a viraltranscription factor originally called EB1, but later calledZEBRA, Z or Zta, is essential for the activation of the productivecycle (6). Moreover, expression of EB1 in B cells latently infectedwith EBV is both necessary and sufficient to activate the transcriptionof all of the productive-cycle genes and the replication ofthe viral genome (6, 11, 39).

The functions of the EBV gene products expressed during theproductive cycle have been only partially characterized, andvery little is known for others, such as the EBV BMLF1 earlygene product originally called EB2 (6) but later called Mtaor SM. This nuclear protein was first described as a promiscuoustranscription factor, as it activates transient expression ofthe chloramphenicol acetyltransferase gene when placed underthe control of many different promoters (29). In contrast, anaccumulation of recent experimental results has given rise tothe notion that the EB2 protein is not a transcription factorbut rather has properties reminiscent of an RNA export factor.These properties can be summarized as follows. The EB2 proteininduces the cytoplasmic accumulation of both intronless andintron-containing RNA (3, 10, 45). The EB2 protein also shuttlesbetween the nucleus and the cytoplasm (2, 10, 45). It seemsto carry two adjacent nuclear export signals (NES) (5) and bindsto RNA in vivo (40), although no EB2-specific RNA motifs onpotential EB2 target RNA have been identified so far.

Most of the EBV early and late mRNAs are transcribed from intronlessgenes. However, it is now clearly established that nuclear exportof mRNA is dramatically increased by splicing (31) which leadsto deposition of a multiprotein export complex (EJC), includingREF/Aly, Y14, RNPS1, SRm160, and Mago, 20 to 24 nucleotidesupstream of the exon-exon junction (22, 24, 26, 27). CellularmRNAs generated from intronless genes require an alternativemode to be efficiently exported from the nucleus to the cytoplasm.Some contain specific cis-acting elements and are exported tothe cytoplasm by cellular factors through interaction with mRNA-boundadapters such as REF, SRp20, 9G8, or U2AF, which are targetsfor cellular nuclear RNA export factors such as TAP (7, 18,37). Hence, it is tempting to speculate that EB2 is such a viraladapter involved in the export of intronless early and/or lateEBV mRNA, similar to its herpes simplex virus type 1 (HSV-1)homolog ICP27 (25). If this is true, the EB2 protein is essentialfor the production of EBV infectious virions.

To test this hypothesis, we first generated an EBV mutant inEscherichia coli in which the BMLF1 gene encoding the EB2 proteinhad been deleted, introduced the mutated viral genome into 293cells, and then selected a clonal population called 293-_BMLF1-KO.In order to induce the productive cycle of EBV, these cellshave to be transfected with an expression plasmid encoding EB1(8). Our studies show that such cells produced infectious EBVparticles only when an expression vector coding for the EB2protein was cotransfected concomitantly with the expressionplasmid coding for EB1. In these cells, oriLyt-dependent DNAreplication was greatly enhanced by EB2 expression. We alsoobserved that EB2 induced the cytoplasmic accumulation of asubset of EBV early mRNAs coding for essential proteins implicatedin EBV DNA replication during the productive cycle. The cytoplasmicaccumulation of some late mRNAs is also induced by the EB2 protein.Two herpesvirus homologs of the EB2 protein, the HSV-1 proteinICP27 and the UL69 protein of human cytomegalovirus (HCMV),only partly rescued the phenotype observed with the EBV_BMLF1-KOmutant.

MATERIALS AND METHODS

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Materials and Methods

Plasmids. The following expression plasmids have been previously described.When Flag precedes the name of the protein, the correspondingprotein has been tagged at its N terminus with the Flag epitope,which can be detected by the M2 monoclonal antibody (MAb) (Sigmacatalog no. F3165). pCMV-BZLF1, contains the BZLF1 cDNA encodingthe EB1 protein (32). paacFlagEB2 contains the intronless BSLF2-BMLF1cDNA (4). paacFlagEB2_RXP expresses an EB2 protein with the RXPrepeat deleted (3), and paacFlagEB2_dNES expresses an EB2 proteinwith the two putative NES mutated as described by Chen et al.(5) (the sequence PVSKITFVTLPSPLASLTL between amino acids 218and 236 has been modified to PVSKITF- - -AAALAS- - -). A. Epsteinkindly provided pUL53/54, the HSV-1 ICP27-expressing vector.Briefly, it contains the HSV-1 ClaI/SacI DNA fragment containingthe UL53 and UL54 genes cloned in pBluescript II SK(+) (Stratagene).The expression plasmid for UL69 was kindly provided by T. Stamminger(30). Plasmids used for transfection were prepared by the alkalinelysis method and purified through two CsCl-ethidium bromidegradients.

Recombinant EBV plasmid. To generate a BMLF1 knockout (KO) mutant in the context of theEBV genome, we used our maxi-EBV technology (8). E. coli strainDH10B was transformed with the EBV genome cloned into a mini-F-factorreplicon, termed p2089 (8, 20). p2089 is a maxi-EBV plasmidwhich carries the F factor origin of DNA replication, the chloramphenicolresistance gene, and the gene for the green fluorescent protein(GFP) under the control of the CMV immediate-early promoter/enhancertogether with the hygromycin resistance gene as a selectablemarker in eukaryotic cells. A second plasmid, termed p2650,was introduced into strain DH10B harboring p2089. p2650 encodesa constitutively expressed recA gene to aid homologous recombinationand the red ${gamma}$ gene to allow transformation of linear DNA fragments.p2650 consists of the plasmid backbone of pST76-amp (34), whichcarries a temperature-sensitive origin of DNA replication derivedfrom pSC101 and confers resistance against ampicillin. In orderto generate p2650, a SacII/KpnI fragment derived from pKY102carrying recA (19) was treated with the Klenow fragment of E.coli polymerase I in the presence of deoxynucleoside triphosphatesand ligated into the Ecl136I-cleaved pST76-amp plasmid to yieldplasmid p2423. This plasmid was again linearized with NsiI andtreated with Klenow enzyme to accommodate a Klenow enzyme-treatedHindIII/XhoI fragment from pBAD-ET ${gamma}$ (51) carrying red ${gamma}$ to yieldp2650.

In order to delete the coding region of the BMLF1 gene, a subcloneof the B95.8 EBV genome was generated in E. coli. This plasmid,termed pSM neo/kan (Fig. 1A), contains the BamHI/EcoRI fragment(nucleotide coordinates 79535 to 82925) of the B95.8 strainof EBV, the AlwNI/SspI fragment from pEGFP-C1 (Clontech) harboringthe kanamycin-neomycin resistance cassette, and the BamHI/BamHIS-fragment (nucleotide coordinates 84234 to 87654) of B95.8.Insertion of the kanamycin-neomycin resistance cassette at thisposition removed the BMLF1 coding region from amino acid 1 to378. A PmlI/NheI fragment derived from pSM neo/kan (Fig. 1A)was electroporated into E. coli DH10 carrying p2089 and p2650,and the bacteria were coselected for antibiotic resistance againstchloramphenicol (30 µg/ml) and kanamycin (50 µg/ml)at 42°C. At this temperature, p2650 is lost rapidly, andcolonies were subsequently analyzed by using various restrictionenzymes to confirm correct recombination. One identified clone(p2688.16) was selected, and its plasmid DNA was prepared onCsCl-ethidium bromide gradients. The plasmid DNA was partiallysequenced to confirm the anticipated deletion of the BMLF1 geneand the integrity of the flanking regions.

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FIG. 1. Mutant EBV lacking its early gene BMLF1. (A) Construction of the BMLF1-negative EBV mutant genome by homologous recombination in E. coli. The diagram shows schematically a linearized fragment of the wild-type EBV genome carrying the BMLF1 gene and the same fragment from the EBV_BMLF1-KO mutant, in which most of the BMLF1 sequence has been replaced by the kanamycin resistance gene. (B) The 293-_BMLFI-KO cell clone was used for all of the following experiments. The cells express GFP because the maxi-EBV genome encodes the GFP gene. The cells were visualized by UV light microscopy. (C) Western blot analysis of the EB2 protein and viral early proteins expressed in the 293 cell clone carrying EBV_BMLF1-KO mutant maxi-EBV. Total proteins were prepared from Raji cells (EBV-infected cells) or from 293-_BMLFI-KO cells mock transfected (lane 4), transfected with only an expression vector for the EBV transcription factor EB1 (lanes 1, 2, and 5), or cotransfected with expression vectors for both EB1 and EB2 (lanes 3 and 6). The nitrocellulose filter was probed with a polyclonal serum directed against the EB2 protein (lanes 1 to 3) or a set of MAbs specific for the EBV R transcription factor, the EBV early protein BMRF1 (EA-D), or the EB1 protein.

Selection of stable 293 cell clones and induction of the productive phase of the EBV life cycle. The maxi-EBV plasmid p2688.16 was transfected into 293 cellsas previously described (8, 20) to establish single-cell clonesthat carry the BMLF1-KO maxi-EBV plasmid. The 293 cells wereselected in the presence of 80 µg of hygromycin per mlfor 4 weeks, and single outgrowing clones were expanded. Thecells were called 293-_BMLF1-KO cells. The single-cell cloneswere transiently transfected with Lipofectamine in 6-well clusterplates as described previously (8, 20) with 0.5 µg ofthe expression plasmid p509 (pCMV-BZLF1) (16) encoding EB1 proteinto induce the EBV productive phase. In order to trans-complementthe BMLF1-negative maxi-EBV, 1.0 µg of a second expressionplasmid encoding EB2 under the control of the CMV immediate-earlyenhancer and promoter was cotransfected (pAAC-FlagEB2). Twoto 3 days later, the supernatant was collected and filteredthrough a 0.45-µm-pore-size filter. Infection of Rajicells with the virus stock was carried out as previously described(11, 20). Transient transfections of 293-_BMLF1-KO cells werealso performed by electroporation as described previously (44),with no apparent variability in the results obtained, in termsboth of virus production and protein expression.

Western blot analysis. Cells were lysed in sodium dodecyl sulfate gel sample buffer.The equivalent of 2 x 10⁶ cells was loaded into each lane ofsodium dodecyl sulfate-10% polyacrylamide gels. After electrophoresis,proteins were transferred to nitrocellulose membranes. Membraneswere incubated either with the anti-BZLF1 MAb Z125 or the anti-BRLF1MAb 8C12 (28) or the anti-BMRF1 MAb NCL-EADE31 (Novocastra)or rabbit polyclonal serum directed against the EB2 protein(3). Anti-mouse or anti-rabbit-horseradish peroxidase conjugate(Amersham) was used as a secondary antibody, and immune complexeswere visualized with the ECL reagent (Amersham).

Transient-replication assays. Transient-replication assays were performed with 293-_BMLF1-KOcells by using a plasmid carrying the complete wild-type oriLyt,termed p968.22, which has been described in detail elsewhere(16). p968.22 was transfected either alone; together with plasmidpCMV-EB1, which expresses the EB1 protein; or together withpCMV-EB1 and paacFlagEB2, an expression plasmid for the EB2protein. Two days after transfections, DNA was prepared by theHirt technique, digested with DpnI and BamHI, subjected to electrophoresisthrough a 0.7% agarose gel, transferred to a nylon N+ membrane(Amersham), and probed with random-primer ³²P-labeled pUC19.The replication efficiency of the oriLyt plasmid p968.22 wasquantified by scanning the Southern blot autoradiogram.

RNA extraction and RT-PCR analysis. Transfected cells were washed twice with phosphate-bufferedsaline, and cytoplasmic RNA was extracted and purified on acesium chloride gradient according to published protocols (6).

We performed reverse transcription-PCR (RT-PCR) on 0.5 µgof purified RNA by using the Titan one-tube RT-PCR system (RocheMolecular Biochemicals). The positions of the primers used forRT-PCR are the following, according to the B95-8 EBV strainsequence (1): for BALF2, 5'-GGAAGACTGTGTGTTTGATGTGGTG-3' (positions161726 to 161750) and 5'-TGATAAAGTTGTCCTGGGCTGC-3' (positions161512 to 161533); for BALF5, 5'-AAGAGGCACTGGAGGATGTTGG-3' (positions153763 to 153784) and 5'-AGGGCTACCCTGTGGGCTTTTTG-3' (positions154116 to 154137); for BBLF2/3, 5'-CTCAGATTTCCAGCCCTATGTCC-3'positions (18129 to 118151) and 5'-CCACAAAGCCCAGGATGAACTC-3'(positions 117556 to 117577); for BBLF4, 5'-GCATCCGTGACTATGGCATTAGC-3'(positions 112076 to 112098) and 5'-CGTGAGTTCTTTAGGGCATCCAC-3'(positions 111849 to 111871); for BMRF1, 5'-CCAGACATACGGTCAGTCCATCTC-3'(positions 80881 to 80904) and 5'-TGCTTCACTTTCTTGGGGTGC-3' (positions81071 to 81091); for BSLF1, 5'-AAAACCTTCTGCTACCACATCGC-3' (positions84786 to 84808) and 5'-CAGCCCTATTTATGATTCTGGAGG-3' (positions84343 to 84366); for BLLF1, 5'-TATGGTGGGGTGGTGTAGGTATG-3' (positions89455 to 89477) and 5'-CAGATTACGGCGGTGATTCAAC-3' (positions89731 to 89752); for BALF4, 5'-TCGTGATAGCGTCTTCTGCGTAG-3' (positions156798 to 156820 and 5'-TGAGCAAAACCAGGAGCAAAAG-3' (positions156896 to 156917); and for BCRF1, 5'-CCACAGGTTCCTGCC-3' (positions10031 to 10045) and 5'-GACAGTCGAAAGGGG-3' (positions 10236 to10250). Quantification of the RT-PCR results was done accordingto a method described previously (3). As a control in our RT-PCRexperiments, endogenous expression of the ß-actinmRNA was also evaluated by RT-PCR. Primers 5'-GCTGCGTGTGGCTCCCGAGGAG3'and 5'-ATCTTCATTGTGCTGGGTGCCAG-3' were used in the PCR to amplifya 690-bp DNA fragment corresponding to the ß-actinmRNA.

RESULTS

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Results

Construction of a BMLF1-negative EBV. The complete genome of the prototype EBV strain B95.8 was clonedin E. coli as described previously (8). In order to facilitategenetic manipulation of the so-called maxi-EBV plasmid in theprokaryotic host, a temperature-sensitive plasmid encoding RecAand Red ${gamma}$ was constructed and termed p2650. This plasmid allowsefficient homologous recombination in a recA-negative backgroundsuch as DH10B. In addition, transformation of DH10B with linearDNA fragments is greatly facilitated by the Red ${gamma}$ gene product,which eliminates endogeneous exonuclease activity, similar toa recently published strategy (51). To introduce the KO of BMLF1,the plasmid pSM neo/kan was cleaved by two restriction enzymes(PmlI and NheI) to accommodate a kanamycin resistance gene flankedby two short stretches of homologous EBV sequences of 82 and76 bp in length (Fig. 1A). The two flanking sequences shouldtarget the kanamycin resistance cassette so that most of thecoding sequence of BMLF1 is deleted by insertional mutation.The linear DNA fragment of pSM neo/kan was introduced into DH10Bharboring the wild-type maxi-EBV p2089 and the temperature-sensitive,RecA- and Red ${gamma}$ -encoding p2650 plasmids. Colonies were selectedon chloramphenicol- and kanamycin-containing plates at 42°C.The maxi-EBV plasmid DNA from single colonies was analyzed withseveral restriction enzymes, and one clone, termed p2866.16,was selected for further analysis. About 1,000 bp covering thedistal parts of the introduced kanamycin resistance cassetteand the flanking EBV DNA was sequenced in order to confirm theoverall composition of the altered locus and the integrity ofthe neighboring genes (data not shown). Large-scale plasmidDNA was prepared from E. coli and purified on CsCl-ethidiumbromide equilibrium density gradients to yield BMLF1-KO maxi-EBVplasmid DNA.

EB2 is essential to produce infectious EBV particles. The production of virus stocks in the maxi-EBV systems requiresthe establishment of a permissive cell line based on 293 cellsthat carry the EBV genome. To establish such a cell line, theBMLF1-KO maxi-EBV plasmid DNA prepared from E. coli was transfectedinto 293 cells, and stable hygromycin-resistant cell clonesthat carry the EBV plasmid extrachromosomally were selected.The correct composition of the maxi-EBV DNA was confirmed bySouthern blot hybridization (data not shown). One cell clone,termed 293-_BMLF1-KO, was used for all of the following experiments.Since maxi-EBV plasmids encode GFP expressed under the controlof the CMV immediate-early promoter and enhancer, the cellswere bright green under UV light (Fig. 1B). Upon induction ofthe productive phase of the EBV life cycle by transient transfectionof an expression vector for the EBV productive-cycle transactivatorEB1 (8), the 293-_BMLF1-KO cells failed to express the EB2 protein(Fig. 1C, lane 2). In contrast, Raji cells transiently transfectedwith a plasmid encoding the EBV transcription factor EB1 readilyexpressed the EB2 protein (Fig. 1C, lane 1). The 293-_BMLF1-KOcells expressed the EB2 protein only when transfected with anexpression plasmid for the EB2 protein (Fig. 1C, lanes 3 and6).

In order to determine the production of progeny virus, the 293-_BMLF1-KOcells were first transiently transfected with an expressionplasmid encoding the EBV transcription factor EB1 to inducethe productive phase of the EBV life cycle. Three days aftertransfection, the cellular supernatant was harvested, centrifuged,and filtered to remove cellular debris. This supernatant wasthen used to incubate Raji cells in order to analyze the presenceof infectious EBV, visualized indirectly by GFP expression inRaji cells as described previously (8, 9, 11, 20). No greenRaji cells could be found when the cells were transfected witha control plasmid (instead of the EB1 expression vector), indicatingthat the 293-_BMLF1-KO cells do not spontaneously produce infectiousviral particles (Fig. 2a). In 293 cells carrying wild-type maxi-EBV,up to 10⁶ infectious particles per ml can usually be detectedin the cellular supernatant. In sharp contrast to this situation,only very few or no green Raji cells could be found in supernatantsfrom EB1-transfected 293-_BMLF1-KO cells, strongly suggestingthat the lack of the BMLF1 gene product causes a very dramaticdecrease in virus production (Fig. 2b). Next, we cotransfectedthe 293-_BMLF1-KO cells with the expression plasmid for the EB1protein together with an expression plasmid for the EB2 protein.As expected, an impressive number of green Raji cells couldbe seen by UV light microscopy, indicating successful transductionof the GFP gene into Raji cells by BMLF1-KO virions (Fig. 2c).The rescue of virus production by cotransfecting an EB2 expressionvector and an EB1 expression vector into 293-_BMLF1-KO cellswas not due to an unusually high expression of the EB1 protein,since its expression level was not altered by concomitant expressionof EB2 (Fig. 1C, compare lanes 5 and 6). Similarly, the expressionlevels of the EBV transcription factor R or the early proteinBMRF1 (EA-D) were not significantly modified by the expressionof EB2 (Fig. 1C, compare lanes 5 and 6). This result suggeststhat the EB2 protein is not required for efficient expressionof these EBV proteins.

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FIG. 2. Infection of Raji cells with different EBV stocks as detected by GFP expression. Raji cells (10⁵) were incubated with 1 ml of supernatant from 293-_BMLF1-KO cells (a) or with the same cells transfected with an expression plasmid encoding the EB1 protein (b) or transfected with expression plasmids encoding both the EB1 and EB2 proteins (c). GFP fluorescence (corresponding to successful infections) was detected 72 h after incubation with the supernatants as described in the text.

These results clearly demonstrated that the EBV BMLF1 gene isessential for the production of infectious EBV particles andthat the disruption of the BMLF1 gene can be functionally trans-complementedby transient expression of its product, the EB2 protein.

The EB2 protein strongly enhances oriLyt-dependent DNA replication in 293-_BMLF1-KO cells. In order to define the molecular level at which the productivecycle of EBV is affected in 293-_BMLF1-KO cells, we analyzedthe efficiency of the viral DNA synthesis. The 293-_BMLF1-KOcells were transfected with the plasmid p968.22, encompassingoriLyt together with an expression plasmid for the EB1 proteinso as to activate EBV's lytic, productive cycle. The plasmidp968.22 did not replicate in the absence of the productive cycle(Fig. 3, lane 1). Upon induction of the productive cycle bytransfection of the EB1 expression vector, p968.22 replicatedin 293-_BMLF1-KO cells, although with low efficiency (Fig. 3,lane 2). Coexpression of the EB2 protein in these cells greatlyenhanced the signal of newly replicated oriLyt DNA (Fig. 3,lane 3), demonstrating that the EB2 protein is not absolutelyrequired for oriLyt-dependent replication but enhances its efficiency.These results also showed that 293-_BMLF1-KO cells are able toreplicate viral DNA in the absence of EB2 but are severely impairedin the production of virus progeny.

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FIG. 3. Enhancement of oriLyt-dependent DNA replication by the EB2 protein. 293-_BMLF1-KO cells were transiently cotransfected with the oriLyt plasmid p968.22 together with a control vector (lane 1), an EB1 expression plasmid (lane 2), or the EB1 and EB2 expression plasmids (lane 3). Newly replicated oriLyt plasmid DNA that was detected by Southern blot hybridization with a pUC probe after digestion of the Hirt DNA with BamHI and DpnI is indicated by an arrow.

EB2 increases the nuclear export of specific EBV early mRNA. Even in the absence of EB2, oriLyt-dependent DNA replicationwas detectable in 293-_BMLF1-KO cells induced by EB1. This resultsuggested that all mRNAs encoding the proteins implicated inDNA replication were transcribed and translated. Since the EB2protein enhanced oriLyt-dependent replication considerably,we speculated that EB2 might have an important effect on thelevel of expression of some of the proteins involved in oriLyt-dependentDNA replication, with the exception of the EA-D proteins (BMRF1),which were not affected by the lack of EB2 (Fig. 1C). SinceEB2 had an effect on the level of lytic DNA replication, EB2function could be involved in the expression of other factors,presumably at the level of their mRNA export. To test this hypothesis,we evaluated by semiquantitative RT-PCR the cytoplasmic accumulationof the early mRNAs coding for the proteins necessary for oriLyt-dependentreplication. The results presented in Fig. 4A provided the followingevidence. (i) The amounts of ß-actin mRNA detectedby RT-PCR in latently infected or induced 293-_BMLF1-KO cellswere comparable in the absence or presence of EB2 (Fig. 4A,lanes 1 to 3). (ii) As expected, the expression level of thelatent gene BYRF1 (EBNA2) was not altered in the latent or productivecycle even when the EB2 protein was expressed (Fig. 4A, lanes7 to 9). (iii) Expression of the BKRF1 gene (EBNA1) has beenshown previously to be increased by the induction of the productivecycle (50). Accordingly, BKRF1 expression was detectable inlatently infected cells (Fig. 4A, lane 4), but its steady-stateexpression level was elevated in the 293-_BMLF1-KO cells uponinduction of the productive cycle by the EB1 expression plasmid(Fig. 4A, lanes 4 and 5). Cotransfection of the EB2 expressionplasmid did not alter this level of the BKRF1 RNA (Fig. 4A,lane 6). These results demonstrated that it is possible to detectvariations in the level of expression of EBV genes by RT-PCRand that the RT-PCR conditions used are suitable for quantitation.

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FIG. 4. The EB2 protein induces the accumulation of specific mRNAs coding for EBV replication proteins. (A and B) RNAs from 293-_BMLF1-KO cells transiently transfected with a control vector or expression plasmids coding for proteins, as indicated, were subjected to RT-PCR analysis with specific primer pairs to detect the ß-actin cellular mRNA and EBV latent mRNA encoding BKRF1 or BYRF1 (A) or the product of the early genes encoding the EBV replication proteins BALF2, BALF5, BBLF2/3, BBLF4, BMRF1, and BSLF1 (B). The PCR products were loaded onto a 2% agarose gel and visualized by ethidium bromide staining. (C) Quantification of five independent RT-PCR assays. The results are presented as the relative cytoplasmic accumulation of the early mRNA in the cells transfected with expression vectors for EB1 and EB2 compared to their accumulation in the cells transfected only with an EB1 expression vector. Error bars indicate standard deviations.

During latency the genes coding for the proteins necessary forDNA replication are not expressed, and their corresponding mRNAswere not detectable by RT-PCR (Fig. 4B, lanes 1, 4, 7, 10, 13,and 16). The faint bands observed lanes 10 and 16 of Fig. 4Bare probably the result of some spontaneous reactivation ofthe productive cycle in the cell culture. When the productivecycle was induced by transfection of the EB1 expression vectorinto 293-_BMLF1-KO cells, all mRNAs from the viral genes involvedin DNA replication could be detected by RT-PCR (Fig. 4B, lanes2, 5, 8, 11, 14, and 17). This result was expected because underthe conditions described above, oriLyt-dependent replicationtook place, although at a somewhat reduced level (Fig. 3). Sincethe EB2 protein enhanced oriLyt-dependent DNA replication, weexpected that EB2 would increase cytoplasmic RNA accumulationfor most of the genes encoding the replication proteins. Unexpectedly,the EB2 protein increased the cytoplasmic accumulation of onlya subset of mRNAs corresponding to the BALF2 gene (single-stranded-DNA-bindingprotein) (Fig. 4B, compare lane 2 with lane 3; see quantificationof five independent assays in Fig. 4C) and the BALF5 gene (DNApolymerase) (Fig. 4B, compare lane 5 with lane 6; see quantificationof five independent assays in Fig. 4C). Cytoplasmic accumulationof the mRNAs of BBLF2/3, BBLF4, BMRF1, and BSLF1 was not apparentlyaffected by the EB2 protein (Fig. 4B; see quantification offive independent assays in Fig. 4C). Taken together, these resultsstrongly suggest that the EB2 protein specifically exports twoout of six mRNAs coding for the viral proteins necessary fororiLyt-dependent DNA replication, which implies that EB2 isnot a nonspecific viral mRNA export factor.

EB2 also induces the cytoplasmic accumulation of some late mRNAs. It has been shown that the HSV infected protein 27 (ICP27) isrequired for the expression of certain early viral proteinsand many late proteins during productive infection. For example,expression of glycoprotein C is severely restricted at the mRNAlevel in the absence of ICP27 (21). In order to determine whetherthe EB2 protein was also required for some late mRNA export,we analyzed the cytoplasmic accumulation of three late mRNAs(BALF4, BLLF1, and BCRF1) by RT-PCR as described above. Thequantified results are presented in Fig. 5, and they show thatthe EB2 protein induced cytoplasmic accumulation of the threemRNAs tested. For these three mRNAs, we observed a twofold increaseof the amount of mRNA accumulation in the presence of EB2 protein,as for some early mRNAs such as BALF5. However, since the replicationefficiency is affected by the absence of the EB2 protein, wecannot exclude the possibility that the results observed withthe late mRNAs were not a consequence of the defect in replicationefficiency.

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FIG. 5. The EB2 protein also induces the cytoplasmic accumulation of late mRNA. RNAs from 293-_BMLF1-KO cells transiently transfected with a control vector or expression plasmids coding for EB1 and EB2 proteins, as described Fig. 4, were subjected to RT-PCR analysis with specific primer pairs to detect the early mRNA BALF5 or the late mRNAs BALF4, BLLF1, and BCRF1. Quantification of three independent RT-PCR assays is shown. The results are presented as the relative cytoplasmic accumulation of the early mRNA in the cells transfected with expression vectors for EB1 and EB2 compared to their accumulation in the cells transfected only with an EB1 expression vector.

trans-complementation of EBV_BMLF1-KO virus by EB2 mutants and functional homologs of other herpesviruses. The EB2 protein contains an Arg-X-Pro tripeptide repeat similarto that described as an RNA-binding motif in the HSV-1 US11protein (38). The EB2 motif also binds RNA in vitro, but deletionof this particular motif in the EB2 protein does not affectits ability to export nuclear RNA in a transient-expressionassay (3). Hence, we wanted to evaluate the effect of this specificdeletion in the EB2 protein in the oriLyt replication assayin 293-_BMLF1-KO cells. The results show that the mutant EB2_RXPprotein is as efficient as the wild-type EB2 protein in enhancingoriLyt-dependent replication (Fig. 6A, compare bars 3 and 4),demonstrating that the Arg-X-Pro motif in EB2 does not supportan essential function in this context. This result was alsoconfirmed by the fact that the EB2_RXP protein was as efficientas the wild-type EB2 protein in its capacity to trans-complementthe EBV_BMLF1-KO virus for the production of infectious virions(data not shown).

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FIG. 6. DNA replication analysis and production of infectious particles in 293-_BMLF1-KO cells trans-complemented with various EB2 mutants and herpesvirus functional homologs. (A) Enhancement of oriLyt-dependent replication by the EB2 protein functional homologs from HSV-1 and HCMV. 293-_BMLF1-KO cells were transiently cotransfected with the oriLyt plasmid p968.22 together with a control vector (bar 1) or expression plasmids coding for proteins as indicated at the bottom of the histogram. The relative replication efficiency of p968.22 in the presence of both EB1 and EB2 was set to 100%. The replication assays were done at least four times, and means and standard deviations are indicated. (B) Infection of Raji cells by various EBV stocks as detected by GFP expression. Raji cells (10⁵) were incubated with 1 ml of supernatant from 293-_BMLF1-KO cells transfected with expression plasmids encoding either the EB1 protein alone (a), the EB1 and EB2 proteins (b), EB1 and a dNES EB2 mutant protein (c), EB1 and ICP27 (d), or EB1 and UL69 (e). GFP fluorescence (corresponding to successful infections) was detected 72 h after incubation with the corresponding supernatant.

The EB2 protein shuttles between the nucleus and the cytoplasmand contains a leucine-rich signal sequence, LPSPLASLTLS, thatmatches the consensus NES identified in the Rev family of proteinsor the HSV-1 ICP27 protein (5). Mutation of this LXL motif alonedid not prevent EB2 shuttling (10), but a double mutation ofthe LXL motif plus an upstream VTL motif was reported to suppressshuttling, suggesting that the EB2 protein has two NES motifs(5). The EB2_dNES mutant protein was still capable of enhancingEB1-induced oriLyt-dependent replication in our transient DNAreplication test, although to a lesser extent than wild-typeEB2 (about a 35% reduction in Fig. 6A, lane 5). The EB2_dNESprotein was also capable of trans-complementing EBV_BMLF1-KOmaxi-EBV for the production of infectious particles but at alower level than the wild-type EB2 protein (Fig. 6B, comparepanel c to panel b). Results from fluorescence-activated cellsorter analyses showed that the supernatant of 293-_BMLF1-KOcells obtained after transient transfection of expression vectorscoding for EB1 and EB2_dNES contained about 1/10 of infectiousvirions compared to supernatants obtained with wild-type EB1and EB2 as assessed by GFP fluorescence of infected Raji cells(data not shown). Our observations suggest that the two NESmotifs might enhance efficient oriLyt-mediated DNA replicationand production of progeny virus. It remains to be seen, however,if shuttling of EB2 will be a prerequisite for both functions.

Different functional homologs of the EB2 protein have been foundin herpesvirus family members. Among them are the ICP27 proteinfrom HSV-1, which has recently been shown to export intronlessviral mRNA in a CRM-1-independent fashion (25), and the UL69protein from HCMV. Hence, we wanted to assess whether ICP27or UL69 proteins could trans-complement BMLF1-deficient EBVfor oriLyt-mediated DNA replication and production of infectiousparticles. The results from the replication assays showed thatICP27 as well as UL69 proteins enhanced EB1-induced oriLyt-dependentreplication, although at a lower level than EB2 protein (Fig.6A, lanes 6 and 7). In sharp contrast, both ICP27 and UL69 proteinsonly very poorly rescued production of infectious EBV particlesin 293-_BMLF1-KO cells upon lytic cycle induction with an EB1expression vector (Fig. 6B, panels d and e). Fluorescence-activatedcell sorter analysis of Raji cells incubated with the supernatantof 293-_BMLF1-KO cells transiently transfected with the expressionvectors for EB1 and ICP27 (or EB1 and EB2 as a control) showedthat the supernatants contained only about 1% of infectiousparticles compared to the wild-type situation. In conclusion,the data show that homologous proteins such as ICP27 or UL69are capable of partially rescuing oriLyt-mediated DNA replication.Unexpectedly, both ICP27 and UL69 were found to be very inferiorin reconstituting virus production in 293-_BMLF1-KO cells.

ICP27 enhances the cytoplasmic accumulation of BALF2 and BALF5 mRNAs. We have shown that the ICP27 protein, like the EB2 protein,enhances oriLyt-dependent replication in 293-_BMLF1-KO cells.Our results also show that the EB2 protein increases the nuclearexport of two early mRNAs, BALF2 and BALF5. In order to discoverwhether the ICP27 protein had the same effect on these mRNAs,we repeated the RT-PCR analysis described above with cytoplasmicRNA derived from 293-_BMLF1-KO cells transfected with an EB1-expressingvector either alone or in conjunction with an EB2- or ICP27-expressingvector. The results show that the EB1 protein alone causes aweak cytoplasmic accumulation of the products of the BALF2 andBALF5 genes (Fig. 7A, lanes 1 and 4; see quantification of fiveindependent assays in Fig. 7B), which is enhanced by coexpressionof the EB2 protein as shown before (Fig. 4 and 7A, lanes 2 and3; see quantification of five independent assays in Fig. 7B).As expected, the ICP27 protein also enhances cytoplasmic mRNAaccumulation of BALF2 and BALF5 transcripts, although the effectis not as profound on the latter (Fig. 7A, compare lanes 2 and5 to lanes 3 and 6; see quantification of five independent assaysin Fig. 7B). Cytoplasmic accumulation of BMRF1 mRNA is not significantlyaffected by the EB2 or ICP27 protein (Fig. 7, lanes 7 to 9;see quantification of five independent assays in Fig. 7B), confirmingour data (Fig. 1C). These observations strongly suggest thatthe ICP27 protein enhances oriLyt-dependent replication in the293-_BMLF1-KO cells by the same mechanism as the EB2 protein.

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FIG. 7. The ICP27 protein induces accumulation of the BALF2 and BALF5 mRNAs. (A) RNAs from 293-_BMLF1-KO cells transiently transfected with expression plasmids encoding proteins as indicated at the top were subjected to RT-PCR analysis with specific primers to detect the product of the early genes encoding the EBV replication proteins BALF2, BALF5, and BMRF1. The PCR products were loaded onto a 2% agarose gel and visualized by ethidium bromide staining following migration. (B) Quantification of three independent RT-PCR assays. The results are presented as the relative cytoplasmic accumulation of the early mRNA in the cells transfected with expression vectors for EB1 and EB2 (gray bars) or EB1 and ICP27 (black bars) compared to their accumulation in the cells transfected only with an EB1 expression vector. Error bars indicate standard deviations.

DISCUSSION

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Discussion

We generated an EBV genome with a deletion of its BMLF1 openreading frame and established a clonal 293 cell line, 293-_BMLF1-KO,that stably carries this EBV mutant genome. As expected, nodetectable EB2 protein is synthesized from this EBV mutant afterinduction of the EBV productive cycle with an EB1-expressingvector. Equally, this EBV_BMLF1-KO cell line is incapable ofproducing infectious progeny virus unless the EB2 protein istransiently expressed in the cells. These observations indicatethat the EB2 protein is a protein essential in the productivephase of the EBV life cycle and also indicates that a cellularprotein cannot substitute EB2's functions during the EBV productivecycle.

Within the family Herpesviridae, a set of homologous polypeptidesthat are implicated in RNA processing and transport has beencharacterized (10, 14, 43). These proteins were initially describedas promiscuous transactivators of viral and cellular gene expression.The most intensively studied member of this group is the posttranscriptionalregulator ICP27 of the alphaherpesvirus HSV-1. ICP27 has beenreported to shuttle between the nucleus and the cytoplasm andto bind intronless viral mRNA but not spliced viral transcripts(42). Recently, a direct role for ICP27 in viral mRNA exporthas been demonstrated (25), and it has been shown that ICP27affects nuclear export of viral RNA via a CRM1-independent pathway(25, 46). ICP27 mutant viruses are defective for virus productionand show reduced levels of viral DNA replication (33, 35, 41).

The ICP27 homolog of the betaherpesvirus HCMV is the multifunctionalphosphoprotein UL69 (48). UL69 is a nuclear protein that isalso able to shuttle between the nucleus and the cytoplasm.Trafficking of UL69 does not involve binding to CRM-1 (30),a property shared by the gammaherpesvirus EB2 protein (10),although other reports suggest the contrary (2). UL69 is alsonecessary for efficient production of HCMV (17).

In this study, we show that in the absence of the EB2 protein,some of the EBV productive-cycle genes are less efficientlyexpressed. It has been shown that the EB2 protein is involvedin the cytoplasmic accumulation of mRNA. Although the EB2 proteinbinds to RNA in vivo and shuttles from the nucleus to the cytoplasm(10, 40), the molecular mechanism by which EB2 acts is not known.

The mechanism of cellular mRNA export from the nucleus to thecytoplasm is partially understood. It has been proposed thatthe splicing machinery positions a protein complex near theexon-exon junction (EJC complex), which interacts with a protein,TAP, that is responsible for targeting the nucleoprotein complexto the nuclear pore complex (26, 27). This mechanism cannottake place for EBV's early and late mRNAs because, in contrastto the majority of the cellular genes, they are transcribedfrom intronless genes. Since the EB2 protein induces cytoplasmicaccumulation of intronless mRNA (3), this suggests that theEB2 protein could be a viral mRNA export factor that links intronlessviral mRNA to the cellular export machinery. As for ICP27, EB2might recruit the cellular export factors REF and TAP to viralmRNA (25), providing viral mRNA with access to the cellularmRNA export pathway. This hypothesis seems to hold true forBALF2 and BALF5 transcripts as indicated by our results. Sinceonly a smaller subset of early viral mRNA appeared to be sensitiveto the action of the EB2 protein, yet another, and presumablydifferent, mechanism is probably involved in nuclear exportof the majority of unspliced EBV mRNA. Cytoplasmic accumulationof spliced viral mRNA such as transcripts encoding the primase-associatedfactor BBLF2/3 was not affected by the EB2 protein, presumablybecause this mRNA transcript is exported by a splicing-dependentmechanism similar to cellular mRNA.

It is clear from our results and those reported by Fixman andcoauthors (12, 13) that the EB2 protein greatly enhances oriLyt-dependentDNA replication. This phenotype is probably the consequenceof the effect of the EB2 protein on cytoplasmic accumulationof some early viral mRNAs rather than a direct effect on viralDNA synthesis. It has been reported previously that the EB2protein can lead to increased cytoplasmic accumulation of fiveout of six EBV core replication proteins in a transient-transfectionassay (45). With our genetic system we could show that the EB2protein significantly increased cytoplasmic accumulation ofonly two EBV core replication proteins, the BALF2 and the BALF5gene products. Thus, the EB2 protein has a selective effecton unspliced viral mRNA. The differences between our resultsand those published by Semmes and coauthors (45) are probablydue to the fact that the viral genes have been expressed fromtransfected expression plasmids and thus the corresponding RNAswere not authentically processed (3). By RT-PCR analysis, weobserved that the cytoplasmic accumulation of mRNAs from a fewlate viral genes was also affected (data not shown), but theircorresponding mRNAs were also readily detectable in the absenceof the EB2 protein. Differences in the level of expression ofsome specific early and late proteins were also observed inthe case of an ICP27 mutant HSV-1 (33).

It has been shown that the HCMV protein UL69, which is a functionalhomolog of the EB2 protein, is packaged into virus particles(17, 49). Although UL69 is a component of the virion, it isdispensable for production of infectious progeny virus (17).Given this report, it appears rather unlikely that cells harboringthe BMLF1-KO EBV genome release noninfectious viral particlesin the absence of EB2. However, this assumption is supportedby our trans-complementation system, in which both ICP27 andUL69 proteins, the two EB2 functional homologs in HSV-1 andHCMV, are capable of supporting oriLyt-dependent DNA replicationin the genetic background of an EB2-defective virus but onlyvery poorly support virion production. Efficient DNA replicationof oriLyt is most likely the consequence of efficient mRNA exportof viral replication factors, which can be mediated by UL69,ICP27, or BMLF1 similarly. In contrast, UL69 or ICP27 proteinswere not able to support all of the functions of the EB2 proteinin terms of production of infectious progeny EBV particles.Since virus production and maturation are very complex processes,it appears likely that one or more viral gene products thatare limiting in the morphogenesis of EBV particles are alsoaffected by the absence of EB2. As an example, the product ofthe BALF4 gene is such a candidate (data to be published elsewhere).In summary, our data strongly suggest that EB2 is responsibleand indispensable for the export of selected, specific viralmRNAs from the nucleus to the cytoplasm, which could constitutea virus-specific pathway of potential interest for targetedantiviral strategies.

ACKNOWLEDGMENTS

The expression plasmids for the ICP27 and UL69 proteins werekindly provided by A. Epstein and T. Stamminger, respectively.I. Mikaélian provided plasmid pSM neo/kan. We thank R.Buckland for reading the manuscript.

This work was supported by the Sonderforschungsbereich 455 ofthe Deutsche Forschungsgemeinschaft to W.H. Research in thelaboratory is supported by INSERM and the Association pour laRecherche contre le Cancer (ARC no. 4357 to A.S.). A.S. andE.M. are CNRS scientists.

FOOTNOTES

* Corresponding author. Mailing address: Laboratoire de Virologie Humaine, INSERM U412, ENS-Lyon, 46 Allée d'Italie, F-69364 Lyon Cedex 07, France. Phone: 33 (0) 4 72 72 81 76. Fax: 33 (0) 4 72 72 87 77. E-mail: hgruffat{at}ens-lyon.fr.

REFERENCES

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