JOURNAL OF VIROLOGY, Nov. 1989, p. 4938-4944 Vol. 63, No. 110022-538X/89/114938-07$02.00/0Copyright C) 1989, American Society for Microbiology

Relative Rates of RNA Synthesis across the Genome ofEpstein-Barr Virus Are Highest near oriP and oriLyt

STAN METZENBERGtMcArdle Laboratory for Cancer Research, University of Wisconsin-Madison,

Madison, Wisconsin 53706

Received 13 March 1989/Accepted 22 July 1989

The rates of Epstein-Barr virus transcription were measured in isolated nuclei from marmoset and humanlymphoblasts transformed in vitro. In B95-8, a marmoset B-lymphoid cell line, the most frequently transcribedviral genes are the EBERs (small nuclear RNAs) and BHLF-1 (encoding a lytic-phase gene product). TheEBERs and BHLF-1 genes are separated by nearly 50 kilobase pairs on the Epstein-Barr virus genome and lieadjacent to (<300 base pairs from) oriP and oriLyt, respectively. oriP and oriLyt are putative origins of viralDNA replication, and each is associated with a transcriptional enhancer element. Among the humanB-lymphoblastoid cell lines tested, only the transcription of EBERs predominates.

Epstein-Barr virus (EBV) is a herpesvirus that can infecthuman B lymphocytes latently (reviewed in references 27,46, and 50). After infection with the virus, B cells areefficiently transformed to a state of indefinite proliferation,and multiple copies of the viral DNA are maintained withinthe nuclei as supercoiled plasmid molecules. The percentageof transformed lymphoblasts that spontaneously enter astate of lytic viral replication (resulting in the release ofvirus) varies between different cell clones and may rangefrom less than 0.001% to greater than 1% per day in culture.In the case of B95-8, an EBV-transformed marmoset cell line(34), the percentage is approximately 5% per day. The viralgenes that can induce lytic growth in B lymphoblasts havebeen identified in recent years (7-9, 11, 12, 16, 19, 30, 35, 43,49, 55), but the causes of the natural variability of entry intolytic replication are unknown.During latency, the cis-acting DNA sequence element oriP

(57) and the trans-acting protein factor EBNA-1 (31, 58)appear to be necessary and sufficient for plasmid replication.This replication of the viral DNA depends upon the cellularDNA polymerase (44) and appears to occur only once percell cycle (18). By contrast, the replication of the viral DNAduring the lytic cycle is mediated by oriLyt (17) and the viralDNA polymerase (59) and results in a 100- to 1,000-foldamplification of the viral genome.Both of the putative origins of viral DNA replication, oriP

and oriLyt, are associated with the activity of transcriptionalenhancers. oriP consists of two necessary components: afamily of tandem 30-base-pair repeats and a dyad symmetryelement (41), both of which are bound by the EBNA-1protein (38). In the presence of EBNA-1, the family ofrepeats is able to serve as a transcriptional enhancer (40),and at least one nearby promoter (5) is known to be sensitiveto its action (48). A transcriptional enhancer has beenidentified as a necessary element of oriLyt, and it appears tobe transactivated by the BRLF1 gene of EBV (7, 8, 17).The expression of viral RNA differs during the latent and

lytic states of EBV infection. In latently infected cells, atleast eight viral genes are expressed, and most of the viraltranscripts are extensively spliced from large overlapping

t Present address: Intercampus Program in Molecular Parasitol-ogy, University of California, Laurel Heights Suite 150, Box 1204,San Francisco, CA 94143-1204.

primary transcripts (reviewed in references 27, 46, and 50).Approximately 50 to 100 genes are expressed from allregions of the viral genome during lytic growth (4, 21, 54).The lytic-state RNAs that accumulate to the highest levelsduring lytic growth are encoded by the BamHI fragments(Fig. 1A) H, L, D, M, b, K, Nhet, I, A, F, and W, listed indecreasing order of their levels of RNA accumulation (21).The small EBER transcripts of EBV accumulate to thehighest levels of any EBV-encoded RNA during latency (1,42) and are also readily detectable during lytic growth (53).The EBER transcripts are each approximately 170 bases inlength (1) and are synthesized by RNA polymerase III (22).Their function during EBV infection is not known, thoughthey can complement a deficiency of virus-associated RNAsduring adenovirus infection (3) and they bind to the Laantigen (15, 29).Although the relative levels of accumulation of EBV-

specific transcripts have been measured (14, 20, 21, 23, 25,42, 51, 52, 54), the relative rates of their synthesis have notbeen studied. Since the work of Carneiro and Schibler (6)and others (reviewed in S. W. Peltz, G. Brewer, P. Bern-stein, R. Kratzke, and J. Ross, Crit. Rev. Biochem., inpress) has indicated that there is no correlation between thelevels of accumulation of many cellular transcripts and theirrates of synthesis, the transcription rates of EBV RNAscannot be predicted from their steady-state levels. For thisreason, the relative rates of synthesis of EBV-specific RNAwere measured in this study by analysis of the products ofnuclear run-on transcription. The most frequently tran-scribed regions of the EBV genome in human lymphoblas-toid cells are the EBER genes, and in B95-8 cells, both theEBERs and BHLF-1 genes are predominant sites of tran-scription. These two frequently transcribed regions liewithin 300 bases of oriP and oriLyt, respectively.

Nuclei used in the preparation of run-on transcripts wereisolated by the method of McKnight and Palmiter (32) andwere stored at -70°C for up to 5 months prior to their use.The cell lines used in this study included B95-8, a marmosetlymphoblastoid cell line (34); Ramos, an EBV-negativeBurkitt's lymphoma cell line (26); and three cloned humanlymphoblastoid cell lines (11/17-5, 11/17-4, and 3/15-31)transformed in vitro with the B95-8 strain of EBV andpassaged less than 6 months in culture (47). The cell lineLCL721, kindly provided by Paula Kavathas and Robert

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FIG. 1. Analysis of nuclear run-on transcripts generated in lymphoblastoid cell lines. (A) Map of EBV genome (thick line), detailing theBamHI restriction map (fragments are given letter names), the scale of nucleotides in kilobase pairs, the locations of oriP and oriLyt (verticalarrows), and EBV DNA inserts contained in recombinant plasmids 1 through 20 (see Table 1). The dashed lines in plasmids 3 and 4 indicateregions of EBV homologous to (but not contained within) the EBV DNA insert. (B) Dot blot hybridization analysis of [32P]RNA probes versus

plasmid targets 1 through 21 (see Table 1). Each spot was prepared by blotting 5 ,ug of linearized plasmid DNA onto a ZetaProbe membrane.The top four rows of plasmid spots were hybridized, as described in the text, to [32P]RNA, prepared from nuclei of the EBV+ cell lines B95-8,3/15-31, 11/17-4, and 11/17-5. Row S (Ramos) was hybridized to a probe generated from EBV- Ramos nuclei. The probe used in row 6(+NaOH) was identical to row 4 (11/17-5), with the exception that the 11/17-5 RNA probe was hydrolyzed (0.5 M NaOH for 1 h at room

temperature, then neutralized) prior to hybridization.

DeMars (24), was also transformed in vitro with B95-8-derived EBV but had been grown in culture for several years

before it was used in these experiments. The cell lines B95-8,3/15-31, 11/17-4, 11/17-5, and LCL721 were found by dot blothybridization experiments to contain an average of 1,000 to2,000, 500 to 1,000, 150, 20, and 5 copies of the viral genome,respectively (data not shown). By indirect immunofluores-cence studies with a human antiserum, the percentages ofviral capsid antigen-positive (VCA+) cells in each cell linewere found to be 5, 2, 1, 0.1, and <0.1%, respectively. Therelease of infectious virus from the human lymphoblastoidcell lines 3/15-31 and 11/17-5 is relatively low, however, withonly 0.0005% and <0.003% of the population releasing virusper 24 h, as compared with a 4% rate of virus release withB95-8 (47). The discrepancy in the human lymphoblastoidcell lines between percent VCA+ and percent virus-releasingcells may indicate that the release of infectious virus isinefficient from these cells (33). Radiolabeled RNAs were

generated in a reaction containing 108 nuclei, [32P]UTP(specific activity, 255 Ci/mmol), and other components, as

described by McKnight and Palmiter (32). After a 1-hsynthetic reaction (the rate of nucleoside incorporation was

linear during this period), the elongated nascent RNAs were

purified by the method of Smith et al. (45) and hybridized toplasmid DNAs affixed (39) to ZetaProbe (Bio-Rad) nylonmembranes by the method of Church and Gilbert (10). Inanalyses that compared run-on RNAs from different celllines, hybridizations were performed identically by usingequivalent amounts of probe prepared in side-by-side reac-

tions (for example, in Fig. 1B, 3 x 106 cpm [32P]RNA fromeach lymphoblast line was hybridized in 0.5 ml of buffer [10]for 12 h at 68°C).

In Fig. 1 are shown the results of the hybridization ofnuclear run-on RNAs, prepared from five lymphoblastoidcell lines, to identical dot blot strips containing 21 plasmids.The 20 plasmids that carry EBV DNA fragments (listed inFig. 1 and Table 1 by index numbers 1 through 20) span theEBV genome, which is 172,282 base pairs in length (2).Plasmid 21 serves as a control for non-EBV-specific hybrid-ization, since it contains only pBR322 sequences and a

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TABLE 1. Description of recombinant plasmids used in these experiments

No. Name Size (kbp) EBV bases included Reference

1 pBamC (EBERs) 14 3994-13215 572 p153.7 (EBERs) 8.1 6285-95193 pBamW2 11 13215-19359a4 pBamCAJW2YHFSalG 25 7315-13734a, 41382-56083 17S p352 (EBNA2 orf) 4.2 48865-499096 pBamHF (BHLF-1) 18 48848-622497 p528 (BHLF-1) 7.9 52325-56081 178 pBamFQU 19 54853-694109 pBamQUPO 20 62249-7783510 pClaC 25 67851-88248 5711 pHindLHI 17 79083-91821 5712 pHindE 16 91821-102891 5713 p365 (EBNA3 orf) 3.9 93033-93434, 93234-9402814 pBamZRSalF 16 101949-113284 5715 p349 (EBNA1 orf) 4.3 107970-108220, 108930-10989216 pHindC 31 110942-136912 5717 pClaD 20 133436-148543 5718 pHindD 24 146916-166481 5719 pBamNhet 14 1666163955b20 p345 (LMP orf) 4.1 167487-168478, 169397-16942321 p48.30 (pBR322+HSV TK) 5.1 None22 p561 (BHLF-1) 3.8 52380-52950, 53210-53820 17

a Homology to 12001-47643.b Spans terminal repeats.

thymidine kinase gene from herpes simplex virus type 1.Hybridization to the EBV-containing plasmids is not detect-able with RNAs prepared from the EBV-negative Burkitt'slymphoma cell line Ramos (Fig. 1B), indicating that thehybridization signals detected with the EBV-positive lym-phoblastoid cell lines B95-8, 3/15-31, 11/17-4, and 11/17-5(Fig. 1B) are likely to be EBV specific. In addition, pretreat-ment of the 11/17-5 probe with alkali abrogates the signals(Fig. 1B), indicating that DNA in the probe is not responsiblefor the hybridization signal.The relative rates of transcription corresponding to each

EBV-carrying plasmid were determined quantitatively fromFig. 1 by laser densitometry of autoradiographic exposuresspanning the range from 40 min to 88 h. The relative rateswere normalized to the number of base pairs of EBV DNAhomologous to each plasmid and, therefore, represent a

transcription rate per EBV base (Table 2).As shown in Table 2, the most frequently transcribed

EBV-specific RNAs in the human lymphoblastoid cell linestested appear to be the EBER genes that lie adjacent to oriP(detected with plasmids 1 and 2, in cell lines 3/15-31, 11/17-4,and 11/17-5, and 11/17-3 [not shown]). In B95-8 cells, EBERtranscription is a predominant signal and is exceeded only bytranscription specific to plasmid 7 (discussed below). In Fig.2A, run-on RNA probes against a Southern blot of p153.7fragments (plasmid 2) were used. From this analysis, it isevident that the frequently transcribed region is indeedlocalized to the EBER genes, rather than to their flankingsequences (for example, it is localized to the 0.65-kilobasepair [kbp] AccI and 5.4-kbp ApaI fragments, rather than the3.4-kbp NsiI-EcoRI, 7- and 0.5-kbp AccI, or 1.2- and 0.87-kbp ApaI fragments). Run-on RNA probes from three celllines were tested, as shown in Fig. 2A, and it is clear that therate of transcription of EBERs increases monotonically withthe average genome copy number (as discussed earlier,B95-8, 11/17-4, and LCL721 contain an average of 1,000 to2,000, 150, and 5 copies of the viral genome per cell,respectively). It is evident that the two EBER transcripts,

EBER1 and EBER2 (also known as Jl and J2 [22]), are notsynthesized at equivalent rates. The EBER1 and EBER2transcripts, each of which is approximately 170 bases inlength, are distinguished by their hybridization (Fig. 2) to1.3-kbp and 0.5-kbp NsiI-EcoRI fragments, respectively,and the rate of EBER1 transcription exceeds that of EBER2in B95-8 cells by approximately threefold. In a cell-freetranscription system from KB cells, Jat and Arrand found

TABLE 2. Relative rates of RNA synthesis in regionsof the EBV genome

Plasmid Lymphoblastoid cell lines testedbno.' B95-8 3/15-31 11/17-4 11/17-5

1 40 20 10 22 90 150 60 103 2 6 0.9 0.34 20 10 0.8 0.25 9 9 3 <16 50 8 2 17 210 9 1 0.88 7 2 1 0.59 3 1 0.3 0.310 4 2 0.6 0.211 7 2 0.9 0.212 6 0.5 0.5 0.313 0.8 <0.8 <0.8 <0.814 3 1 0.5 0.215 <0.8 <0.8 <0.8 <0.816 6 3 0.6 0.217 1 1 0.5 0.118 8 3 1 0.519 2 4 0.4 0.120 5 5 2 <1

a Identified in Table 1.b Data represent levels of run-on RNA detected with each plasmid in Fig. 1,

divided by the number of base pairs of the EBV sequence that are homologousto each plasmid target. Units are arbitrary. Use of < symbol indicates belowlimit of detection.

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FIG. 2. Analysis of lymphoblast run-on transcripts specific tooriP region. (A) A 2-,ug portion of p153.7 DNA (plasmid 2) wasdigested with NsiI and EcoRI (abbreviated N + E; lanes 1, 4, and 7),AccI (abbreviated Ac; lanes 2, 5, and 8), or ApaI (abbreviated Ap;lanes 3, 6, and 9), separated on a 1.2% agarose gel by electropho-resis, and blotted to a ZetaProbe membrane. Lanes 1 though 3, 4through 6, and 7 through 9 of the blot were hybridized, respectively,to equal amounts (the same number of counts per minute, the samespecific activity) of nuclear run-on RNA probes prepared fromB95-8, 11/17-4, or LCL721 nuclei. To the left, the electrophoreticmobilities of fragments of phage A restricted with BstEII are shownand correspond from top to bottom with the gel origin and fragmentsof length 8.4, 7.2, 6.3, 5.7, 4.8, 4.3, 3.7, 2.3, 1.9, 1.37, 1.26, and 0.70kbp. The sizes (in kilobase pairs) of salient plasmid bands areindicated with arrows. (B) Restriction map of plasmid p153.7 andstructure of EBV DNA insert. In the top map, the wavy lineindicates non-EBV sequences from the vector and the thick lineindicates the EBV DNA insert (kilobase pair scale at bottom). Thelocation of the EBER1 and EBER2 genes are indicated (arrows), asare the repeat and dyad structures of oriP (bars). The three restric-

the opposite result (22); they found that EBER2 transcrip-tion far exceeded that of EBERL. The ratio of EBERl/EBER2 transcription could clearly depend upon the cell lineused in an experiment or on a variety of other experimentaldetails.

Aside from the transcription of EBER genes, the tran-scription specific to plasmids 4, 6, and 7 appears to be apredominant signal in B95-8 (Fig. 1; Table 2). On the basis ofprevious analyses of the steady-state levels of lytic-phasetranscripts, it seemed likely that this signal would be due totranscription of either BHLF-1 or BHRF-1 originating fromthe divergent promoters surrounding oriLyt (see map in Fig.3B) (21, 23, 28, 36, 37, 49). As demonstrated in Fig. 3A, thefrequently transcribed B95-8 RNA specific to the pBamHF(plasmid 6) region hybridizes with the BHLF-1 gene (forexample, the 5.5-kbp BamHI-BglII and 3.7-kbp BamHI-SstII fragments), although approximately 50% of the overalltranscription appears to be from regions upstream of BHLF-1. The transcription of these upstream (non-BHLF-1) se-quences in LCL721 is roughly half of that in B95-8, asdetected by hybridization to the 3.4-, 2.4-, and 1.6-kbpBamHI-BglII fragments and the 6.0-, 2.2-, and 1.3-kbBamHI-SstII fragments of pBamHF.The differences in BHLF-1 transcription between B95-8

and LCL721 cells are 25-fold, on the basis of hybridization tothe 4.4-kbp fragment of p528 (plasmid 7) digested with BglII,and at least 100-fold, on the basis of hybridization to the2.7-kbp fragment of p561 (plasmid 22) digested with AatIIand KpnI (Fig. 3A). This is consistent with the observationthat the percentage of VCA+ cells is at least 50-fold higher inB95-8 than in LCL721 and that BHLF-1 is expressed earlyafter induction of the lytic state (23, 49). Nonetheless, theexpression of BHLF-1 is not strictly proportional to thepercentage of VCA+ cells in 3/15-31 or 11/17-4. The rate oftranscription specific to p528 (plasmid 7) is 20-fold higher inB95-8 than in 3/15-31 (see Table 2), yet the difference inpercentage of VCA+ cells (5 versus 2%) is less than 3-fold.Since B95-8 is a marmoset cell line and the other lympho-blastoid cell lines are human, this disproportionality couldreflect a species-specific difference in the use of the BHLF-1promoter or some other fundamental difference in the celllines.The rates of RNA synthesis detected with the 20 EBV-

containing plasmids decrease across the panel of cell linesB95-8, 3/15-31, 11/17-4, and 11/17-5 (Fig. 1; Table 2). This isalso the order in which the cells decrease in their averageEBV DNA copy number and in their percentages of VCA+cells. One interpretation of these results is that a variablepercentage of cells may have entered the lytic phase of viralgrowth in each cell line, and this lytic subpopulation isresponsible for the bulk of the transcription detected in thisassay. This model is supported by the observation that someof these plasmids (for example, plasmids 16, 17, and 18)detect transcription in regions of the EBV genome notknown to contain latent-phase genes. Small plasmids such asnumbers 2, 5, 13, 15, and 20 are specific DNA fragmentsoverlapping the EBERs, EBNA-2, EBNA-3A, EBNA-1, andLMP genes, respectively. These genes are all expressedduring latent infection (reviewed in references 27, 46, and

tion maps (N + E, Ac, and Ap) correspond to sequential lanes inpart A, and the sizes (in kilobase pairs) of restriction fragments areindicated below each line. The 7.0-kbp AccI and 5.4-kbp ApaIfragments span the EcoRI site at which the map was linearized, andso each is represented in two segments.

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FIG. 3. Analysis of lymphoblast run-on transcripts specific to theoriLyt region. (A) Digested DNAs were separated on a 1.0% agarose

gel by electrophoresis and blotted to a ZetaProbe membrane. A 1jig-portion of pBamHF (plasmid 6) was digested with BglII andBamHI (abbreviated Bg + Ba; lanes 1 and 5) or SstII and BamHI(abbreviated Ss + Ba; lanes 2 and 6). In lanes 3 and 7, 3 jig of p528(plasmid 7) was digested with BgIII (abbreviated Bg), and in lanes 4and 8, 2 jig of p561 (plasmid 22) was digested with AatII and KpnI(abbreviated Aa + Kp). Lanes 1 through 4 and 5 through 8 were

hybridized, respectively, to equal amounts (the same number ofcounts per minute, the same specific activity) of nuclear run-on

RNA probes prepared from B95-8 or LCL721 nuclei. To the left(dashes) are shown the mobilities of BstEII-digested fragments ofphage A (see Fig. 2B legend), and the sizes (in kilobase pairs) ofsalient restriction fragments are indicated with arrows. (B) Restric-tion maps of plasmids pBamHF, p528, and p561, and the structure ofEBV DNA insert. EBV DNA inserts are shown as thick lines andare aligned with the nucleotide scale (in kilobase pairs) at bottom.

50), but only the transcription of the EBER genes is consis-tently detectable in this nuclear run-on analysis. In B95-8cells, the rates of transcription of the EBNA-2, EBNA-1,and EBNA-3 open reading frames (plasmids 5, 13, and 15,respectively) are consistently lower than the overall rates oftranscription in larger regions that encompass these genes(plasmids 4 [or 6], 12, and 14, respectively), as shown inTable 2. A likely interpretation is that plasmids 4, 6, 12, and14 detect the synthesis of lytic-phase transcripts from re-gions adjacent to the latent-phase genes, and these tran-scripts contribute significantly to the overall rates. Since theEBV-specific transcripts that accumulate in human lympho-blastoid cell lines are predominantly latent-phase (reviewedin 46), it is interesting that the relative rates of transcriptionof lytic-phase genes may predominate in this assay. The datacould be explained if the half-lives of lytic-phase transcriptswere relatively shorter than those of latent-phase tran-scripts, or alternatively, if the cell lines previously used inthe characterization of steady-state levels of transcripts hadunusually low rates of spontaneous entry into the lytic stateof viral growth.

In this study it was determined that the EBERs andBHLF-1 genes are the most frequently transcribed genes inB95-8 cells. Since the transcripts for these genes are knownto accumulate to the highest steady-state levels (1, 21, 42),these data suggest that their regulation is predominately atthe level of transcription rather than RNA stability. Thejuxtaposition of these genes with oriP and oriLyt is puzzling.Considering the large size of the viral genome (172 kbp), itseems unlikely that simply by chance the most frequentlytranscribed genes (EBER and BHLF-1) would lie adjacent to(<300 base pairs distant) components of oriP and oriLyt(Fig. 2B and 3B). It is likely that this phenomenon representsan influence of oriP and oriLyt, as transcriptional enhancersor as functional origins of replication, upon transcription ofthe nearby sequences. The association of transcriptionalenhancers and DNA replication origins has been well stud-ied, as recently reviewed by DePamphilis (13), and a numberof viral examples are known. The gene product from BZLF-1 (also known as ZEBRA [16] and EB1 [9]) directly orindirectly induces higher steady-state levels of BHLF-1RNA (23, 49) and directly or indirectly induces oriLytactivity (17), but it is not known whether the replicative andtranscriptional activities of oriLyt are in some way depen-dent upon each other. Similarly, the EBNA-1 gene producttrans activates both oriP (31, 58) and a transcriptionalenhancer contained within oriP (40), and it has not beenpossible (to date) to obtain replication in the absence ofenhancer activity in that system (56). In studies with oriP-containing vectors (57), the presence or absence of theEBER genes had little effect on the efficiency of plasmidmaintenance (though an effect from the EBERs may stillonly be realized in the context of the intact viral genome). Ifthe expression of EBERs depends upon EBNA-1 proteinbinding to oriP, this might prove to be an interesting exampleof a case where a transcriptional enhancer (or adjacent

Vector sequences are indicated by wavy lines. The locations of theopen reading frames BHLF-1, BHRF-1, BFLF-2, BFLF-1, BFRF-1,BFRF-2, and BFRF-3 (nomenclature from reference 2) are indicatedat the top with arrows, and the two identified segments of oriLyt are

indicated by bars. Four restriction maps are shown below, includingtwo that are shown, respectively, above and below the pBamHFplasmid map. Restriction fragment sizes (in kilobase pairs) are

indicated below or above each map. A deletion of an internal KpnIfragment in p561 is indicated by (A) and a gap in the map.

B

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sequence) activates transcription from an RNA polymeraseIII promoter.

I thank Bill Sugden, Jeff Ross, Aida Metzenberg, WolfgangHammerschmidt, Joyce Knutson, Jennifer Martin, and Tim Middle-ton for helpful advice and critical reviews of the manuscript. I thankTerry Stewart for photographic assistance, and Noreen Warren andJulie Breister for technical assistance.

Finally, I thank Dr. Bill Sugden for supporting this research,under Public Health Service grant PO1-CA22443 from the NationalInstitutes of Health.

LITERATURE CITED1. Arrand, J. R., and L. Rymo. 1982. Characterization of the major

Epstein-Barr virus-specific RNA in Burkitt's lymphoma-derivedcells. J. Virol. 41:376-389.

2. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J.Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell,C. Seguin, P. S. Tuffnell, and B. G. Barrell. 1984. DNAsequence and expression of the B95-8 Epstein-Barr virus ge-nome. Nature (London) 310:207-211.

3. Bhat, R. A., and B. Thimmappaya. 1983. Two small RNAsencoded by Epstein-Barr virus can functionally substitute forthe virus-associated RNAs in the lytic growth of adenovirus 5.Proc. Natl. Acad. Sci. USA 80:4789-4793.

4. Biggin, M., M. Bodescot, M. Perricaudet, and P. Farrell. 1987.Epstein-Barr virus gene expression in P3HR1-superinfectedRaji cells. J. Virol. 61:3120-3132.

5. Bodescot, M., M. Perricaudet, and P. J. Farrell. 1987. Apromoter for the highly spliced EBNA family of RNAs ofEpstein-Barr virus. J. Virol. 61:3424-3430.

6. Carneiro, M., and U. Schibler. 1984. Accumulation of rare andmoderately abundant mRNAs in mouse L-cells is mainly post-transcriptionally regulated. J. Mol. Biol. 178:869-880.

7. Chavrier, P., H. Gruffat, A. Chevallier-Greco, M. Buisson, andA. Sergeant. 1989. The Epstein-Barr virus (EBV) early promoterDR contains a cis-acting element responsive to the EBV trans-activator EB1 and an enhancer with constitutive and inducibleactivities. J. Virol. 63:607-614.

8. Chevallier-Greco, A., H. Gruffat, E. Manet, A. Calender, and A.Sergeant. 1989. The Epstein-Barr virus (EBV) DR enhancercontains two functionally different domains: domain A is con-stitutive and cell specific, domain B is transactivated by theEBV early protein R. J. Virol. 63:615-623.

9. Chevallier-Greco, A., E. Manet, P. Chavrier, C. Mosnier, J.Daillie, and A. Sergeant. 1986. Both Epstein-Barr virus (EBV)-encoded trans-acting factors, EB1 and EB2, are required toactivate transcription from an EBV early promoter. EMBO J.5:3243-3249.

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