Epstein-Barr Virus RNA - PubMed Central Canada

JOURNAL OF VIROLOGY, May 1981, p. 649-660

Vol. 38, No. 2

0022-538X/81/050649-12$02.00/0

Epstein-Barr Virus RNA VI. Viral RNA in Restringently and Abortively Infected Raji Cells WALTER KING, VICKY VAN SANTEN, AND ELLIOTT KIEFF* Section of Infectious Disease, Department of Medicine, and Committee on Virology, University of Chicago, Chicago, Illinois 60637

Received 10 October 1980/Accepted 22 January 1981

Nuclear and polyadenylated RNA fractions of Raji cells are encoded by larger fractions of Epstein-Barr virus DNA (35 and 18%, respectively) than encode polyribosomal RNA (10%). Polyribosomal RNA is encoded by DNA mapping at 0.05 x 10" to 0.29 x 108, 0.63 x 108 to 0.66 x 108, and 1.10 x 108 to 0.03 x 108 daltons. An abundant, small (160-base), non-polyadenylated RNA encoded by EcoRI fragment J (0.05 x 10" to 0.07 x 108 daltons) is also present in the cytoplasm of Raji cells. After induction of early antigen in Raji cells, there was a substantial increase in the complexity of viral polyadenylated and polyribosomal RNAs. Thus, nuclear RNA was encoded by 40% of Epstein-Barr virus DNA, and polyadenylated and polyribosomal RNAs were encoded by at least 30% of EpsteinBarr virus DNA. Polyribosomal RNA from induced Raji cells was encoded by Epstein-Barr virus DNAs mapping at 0.05 x 108 to 0.29 x 108, 0.63 x 108 to 0.66 x 108, and 1.10 x 108 to 0.03 x 108 daltons and also by DNAs mapping within the long unique regions of Epstein-Barr virus DNA at 0.39 x 108 to 0.49 x 108, 0.51 x 108 to 0.59 x 108, 0.66 x 108 to 0.77 x 108, and 1.02 x 108 to 1.05 x 108 daltons. The Raji cell line was established over 10 years ago from a culture of an African Burkitt tumor biopsy (43). Raji cells each contain 50 copies of Epstein-Barr virus (EBV) DNA in an episomal, circular form (1, 19, 26, 32, 38, 42). Studies of the restriction endonuclease sites in the viral DNA in Raji cells have indicated that this DNA is heterogenous (19). One form is similar in organization to other EBV DNAs (6, 13-15, 19, 44). A map of the BamHI and EcoRI restriction endonuclease fragments in "standard" Raji DNA is shown in Fig. 1 (19). All map coordinates in this paper are given relative to this Raji DNA map. Other "nonstandard," or defective, forms of viral DNA have been identified in Raji cells in which DNAs mapping at 0.25 x 10" to 0.29 x 108 and 0.39 x 108 to 0.43 x 108 daltons or DNAs mapping at 0.78 x 108 to 0.83 x 108 and 1.10 x 108 to 1.13 x 108 daltons are more closely linked to DNA which maps at 0.59 x 108 to 0.63 x 108d in "standard" DNA (19). Almost every Raji cell contains an intranuclear antigen, EBNA (45), which is probably specified by EBV. Another new antigen, LYDMA, may be present on the cell surface, but has not been well characterized (30, 51). No other virus-associated antigens have been detected in Raji cells or in African Burkitt tumor tissue (33, 45, 46). The word restringent has been used to characterize this restricted state of EBV expression (39). Hybridizations of cellular RNAs

to EBV DNA have indicated that there is extensive transcription of EBV DNA in Raji cells and

other restringently infected cells and that there is selective processing, so that a subset of RNAs encoded by approximately 10% of the EBV DNA accumulates on polyribosomes (18, 27-29, 39, 41, 52). Previous studies of the DNA which encodes viral RNA in restringently infected Raji cells are limited and have yielded discordant results (28, 41, 48). In one series of studies, polyadenylated polyribosomal RNA from Raji celLs hybridized to 20% of EcoRI fragment A (map position, 0.07 x 108 to 0.39 x 108 daltons [28, 41]). These results are compatible with the initial mapping studies of polyadenylated RNAs from another Burkitt tumor cell line (Namalwa) (41) and with more detailed analyses of polyribosomal RNA from a cell line growth transfonned in vitro by EBV (29). However, in another study, radioiodinated cytoplasmic RNA from Raji cells hybridized almost exclusively to EcoRI fragment J (48) (map position, 0.05 x 108 to 0.07 x 108 daltons). Rymo concluded that EcoRI-J encodes the most abundant mRNA(s) in Raji cells (48). If viral RNA in Raji cells is encoded by the same DNA that encodes viral RNA in cells growth transformed by EBV, this would suggest that EBV plays a functional role in Raji cells similar to its functional role in cells known to be growth transforned by EBV. To resolve this question, we used several approaches to map the DNA

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which encodes RNA in restringently infected Raji cells. Our data indicate that three regions of the EBV genome outside EcoRI-J encode abundant polyribosomal RNAs and that the abundant RNA encoded by EcoRI-J is a small, non-polyadenylated RNA. Thus, the RNAs specified by EBV in the Raji Burkitt tumor cell line are similar to the RNAs specified by EBV in the cell line transformed by EBV in vitro (29, 53a). A more permissive phase of EBV infection (abortive infection) can be induced in Raji cells with halogenated pyrimidines, butyrate, or phorbol esters or by superinfection with the P3HR-1 strain of EBV (2, 11, 16, 23, 25, 37, 50, 55). The onset of abortive infection is marked by the appearance of the virus-associated early antigen complex and the cessation of host macromolecular synthesis (12, 22). The immediate significance of the early antigen complex is that early antigen-reactive antibodies are specifically correlated with acute primary EBV infection and with the occurrence of EBV-associated malignancies (21, 22). Polyadenylated RNAs encoded by at least 20% of the EBV DNA are detectable in abortively infected Raji cells (39). A total of

11 polypeptides immunoprecipitate from abortively infected Raji cells with early antigen-reactive human sera (37). There have been no previous attempts to map the DNA which encodes polyribosomal RNA in abortively infected cells. Mapping of these domains is a logical step in the mapping of the specific viral polypeptides associated with the early antigen complex. MATERIALS AND METHODS Cell culture. Cultures of B95-8 and W91 cells were maintained as previously described in complete medium, which consisted of RPMI 1640 medium supplemented with 10% fetal calf serum (both obtained from GIBCO Laboratories) (13, 35, 36). Raji cells were obtained from W. Henle, Childrens Hospital, Philadelphia, Pa., and were maintained in exponential growth by seeding cultures with 105 viable cells per ml of complete medium. For the induction of early antigen, iododeoxyuridine (30 ,ug/ml) (Sigma Chemical Co.) was added to complete medium 24 h after feeding, and the cells were incubated in the dark at 35°C for 3 days; after this, 90% of the medium was removed and replaced with an equal volume of fresh medium. After 3 days, 5 to 10% of the cells were positive for early antigen, and the cultures were harvested for the isolation of RNA. Sera positive for early antigen were from Gilbert Lenoir, International Agency for Re-

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search on Cancer, Lyon, France. Preparation of Raji RNAs. Polyadenylated and non-polyadenylated RNAs were prepared as previously described (29). For most experiments involving nuclear and polyribosomal RNAs, nuclei and cytoplasm were separated by differential centrifugation of Nonidet P-40-treated cells, and polyribosomes were isolated by velocity sedimentation through discontinuous sucrose gradients (18). RNA was extracted by suspending nuclei or polyribosomes in a solution containing 0.005 M EDTA, 0.05 M Tris-hydrochloride (pH 7.4), 0.5% sodium dodecyl sulfate, and 0.3 mg of proteinase K (EM Laboratories) per ml at 64°C and centrifuging the RNA through a cushion of neutral cesium chloride (density, 1.730 g/cm3). Polyribosomal RNA to be used as a template for avian myeloblastosis virus reverse transcriptase was prepared by magnesium precipitation of cytoplasmic ribonucleoprotein complexes (40) in the presence of 0.01 M vanadyl ribonucleoside complex (4). RNA was extracted from magnesium-precipitated polyribosomes as described above, and polyadenylated RNA was separated by oligodeoxythymidylic acid cellulose column chromatography (39). All RNAs were incubated with 50 ug of iodoacetate-treated DNase I (RNase-free; Worthington Diagnostics) per ml and reextracted before hybridization (29, 41). Cytoplasmic RNA for electrophoresis in agarose gels was prepared by suspending cells in a solution containing 0.01 M NaCl, 0.02 M Tris-hydrochloride (pH 7.4), 0.4% Triton X-100, 0.01 M vanadyl ribonucleoside complex, and 2% sucrose for 5 min at 4°C. After removal of nuclei by centrifugation at 3,000 x g for 5 min, the mixture was added to an equal volume of a solution containing 7 M urea, 0.35 M NaCl, 0.01 M Tris-hydrochloride (pH 7.4), and 1% sodium dodecyl sulfate. This solution was extracted with phenol and chloroform, and the RNA was precipitated at -20°C after 2 volumes of ethanol were added. Preparation and hybridization of RNA blots. To determine the size of the Raji RNA homologous to EcoRI fragment J of EBV DNA, 5 jig of polyadenylated cytoplasmic RNA or non-polyadenylated cytoplasmic RNA was denatured at 60°C for 5 min in 20 pl of 50% recrystallized formamide-2.2 M formaldehyde in electrophoresis buffer consisting of 0.02 M morpholinepropanesulfonic acid (Sigma) (pH 7.0), 0.005 M sodium acetate, and 0.001 M EDTA. The samples were run at 60 V for 9 h in a horizontal 0.8% agarose gel (20 by 20 by 0.6 cm) containing 1 ug of ethidium bromide per ml and 2.2 M formaldehyde in electrophoresis buffer. The gel was then soaked in 20x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) for 45 min, and the RNA was blotted onto nitrocellulose paper (Milipore Corp.) by using lOx SSC. The nitrocellulose paper was then baked at 70°C in vacuo for 5 h and prehybridized in a solution containing 50% formamide, 5x SSC, 0.02% (wt/vol) Ficoll, 0.02% (wt/vol) polyvinylpyrolidine, 0.02% (wt/vol) bovine serum albumin (5), 0.02 M sodium phosphate (pH 6.5), 100 ug of denatured salmon sperm DNA per ml, and 0.1% sodium dodecyl sulfate at 50°C for 2 to 4 h. The solution was made 10% in dextran sulfate (54), and 107 cpm of denatured 32P-labeled EcoRI fragment J DNA obtained from pDK 10 (BamHI fragment C)

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(6) was added; this mixture was incubated at 50°C for 15 h. The nitrocellulose blots were washed sequentially in 2x SSC, lx SSC, 0.5x SSC, and O.lx SSC at 720C, dried, and exposed to SB-5 X-ray film at -700C with a DuPont Lightning-Plus intensifying screen. Purification, in vitro labeling, and hybridization of RNA to labeled viral DNA in solution. Extracellular virus was purified from the supernatant media of B95-8 cultures, and viral DNA was extracted as previously described (7, 13). Cloned B95-8 BamHI fragments A, B, C, E, H, I, K, L, 0, V, and X and SalI fragment F were kindly provided by Timothy Dambaugh (6). Cloned B95-8 EcoRI fragment Dhet-IJhet containing fused termini and cloned W91 EcoRI fragments C and F were kindly provided by Nancy RaabTraub and Christopher Beisel (6, 44). DNAs were digested with restriction endonucleases, and the fragments were separated in 0.6% agarose gels. The region of each gel containing the EBV DNA fragment was excised from the gel with a sterile razor blade, placed in dialysis tubing containing 5 ml of running buffer (17), and subjected to electrophoresis at 60 V for 3 h. The DNA was dialyzed extensively against a solution containing 0.01 M Tris-hydrochloride (pH 7.2), 0.001 M EDTA, and DOWEX 50W-X8. The DNA was then precipitated with 2 volumes of ethanol after 0.15 M NaCl was added. Viral DNA was labeled in vitro by using Escherichia coli DNA polymerase I (Boehringer Mannheim Corp.) and [32P]dCTP (500 Ci/mmol; Amersham Corp.) (13). The specific activities of the labeled DNA fragments and the virion DNA were 0.5 x 108 to 0.8 x 108 cpm/,tg and 1.0 x 108 to 1.5 x 108 cpm/tg, respectively. Between 10-2 and 10-3 Lg of 32P-labeled EBV DNA was mixed with 0.5 mg of polyribosomal RNA or nuclear RNA or with 0.05 mg of polyadenylated RNA in 0.05 ml of hybridization buffer (18, 29). Each mixture was divided into 5-1l samples, which were incubated at 110°C for 5 min and then at 72°C for periods up to 24 h. The procedures used for determining the kinetics of RNA hybridization to labeled EBV DNA have been described previously (18, 39). The plotted data reflect the net hybridization of RNA to viral DNA after correction for (i) the S1 nuclease resistance of denatured 32P-labeled EBV DNA (3 to 5%), (ii) the extent of probe renaturation in the presence of nonhomologous yeast RNA (3 to 5% at 18 h), and (iii) the extent to which the probe hybridized to excess denatured viral DNA (75 to 80%) (18, 39). The amount of virus-specific RNA in each preparation was calculated from the rate of hybridization of RNA to labeled DNA (9, 10, 18, 24, 39). The hybridization rate constant was derived from reconstruction experiments by using known amounts of denatured viral DNA (18, 39) and was corrected for repeated sequences, which constitute approximately 20% of EBV DNA (13-15). No correction was made for differences between the rates of hybridization of denatured DNA and RNA (10, 24). For all experiments we included a control, in which alkali-treated RNA had no effect on the rate of renaturation of labeled virus DNA, indicating that viral DNA was not present in the RNA preparations. Identification of viral DNA fragments encoding RNA. Two series of experiments were performed to identify the viral DNA fragments which encoded

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mRNA in Raji cells or in iododeoxyuridine-treated Raji cells. The first method of preparation of labeled viral DNA complementary to infected cellular RNA has been described in detail previously (29, 41) and involved two cycles of hybridizing 32P-labeled EBV DNA to Raji RNAs in solution, digesting at least 80% of the unhybridized labeled DNA with Si nuclease, and separating the DNA-RNA hybrid from residual denatured and native DNAs by isopycnic banding in neutral cesium sulfate. A comparison of the amounts of labeled DNA in the single-stranded DNA regions of the cesium sulfate gradients after the first and second cycles of hybridization indicated that 95 to 98% of the single-stranded DNA which survived Si nuclease treatment was removed by the first cycle of isopycnic banding. Assuming a similar separation of DNA-RNA hybrids from denatured DNA on the second cesium sulfate gradient, as indicated by the banding of added native and denatured Klebsiella pneumoniae marker DNAs, less than 5% of the labeled DNA in the DNARNA hybrid region of the second cesium sulfate gradient of DNA hybridized to polyribosomal RNA and less than 1% of the DNA in the DNA-RNA hybrid region of the second gradient of DNA hybridized to nuclear RNA could be due to contamination with residual denatured DNA (29). After two rounds of this three-step procedure, the 32P-labeled DNA-RNA hybrids in the region of 1.460 to 1.570 g/cm3 (Fig. 2) were pooled, treated with 0.3 M NaOH at 110°C for 10 min, neutralized, and hybridized to Southern blots (49) containing separated HindIII (data not shown), EcoRI, or BamHI fragments of EBV DNA or cloned fragments of EBV DNA. The second method to identify the EBV DNA fragments which encoded RNA in Raji cells was to synthesize DNA complementary to the polyadenylated polyribosomal RNA of Raji cells, using avian myeloblastosis virus reverse transcriptase and [32P]dCTP in an oligodeoxynucleotide-primed reaction (29, 53). The reaction was terminated after 4 h by adding 0.01 M

EDTA and 0.5% sodium dodecyl sulfate. Salmon sperm DNA was added (final concentration, 100,g/ ml), and the mixture was deproteinized with phenol and chloroform. The nucleic acid was precipitated twice by adding 0.15 M NaCl and 2 volumes of ethanol. The RNA was hydrolyzed partially by adding 0.3 M NaOH and incubating the mixture at 110°C for 10 min. The mixture was neutralized and hybridized to Southern blots (49) as described above.

RESULTS Comparison of the complexity and amounts of viral nuclear, polyadenylated and polyribosomal RNAs in restringently and abortively infected Raji cells. The nuclear and polyribosomal RNAs from restringently infected Raji cells hybridize to 35 and 10% of EBV DNA, respectively, as previously reported (18, 39). The abundance of these RNAs (6 and 0.6 x 10-2%) is higher than previously reported, since in previous studies (18, 39) the hybridization rate constant for viral DNA was not corrected for the repeated sequences in EBV DNA (6, 13-15). The polyadenylated RNA from Raji cells is homologous to 18% of EBV DNA. Previous studies (39) in which polyadenylated RNA was treated extensively with DNase before oligodeoxythymidylic acid chromatography yielded lower values for the complexity of polyadenylated RNA from Raji cells as a consequence of degradation of the polyadenylated RNA by residual RNase in the DNase preparations (van Santen et al., in press). After induction, nuclear RNA was homologous to at least 40% of EBV DNA, whereas polyadenylated RNA and polyribosomal RNA were homologous to at least 30% of EBV DNA. 16S0 1600 . I550 ft 1500 E 400

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The amount of viral RNA in the nuclei increased less than twofold after induction, whereas the amounts of viral polyadenylated RNA and polyribosomal RNA increased three- and fivefold, respectively (Fig. 3 and Table 1). Mapping of DNA which encodes RNA in restringently and abortively infected Raji cells. The viral DNA which encodes RNA in Raji cells was mapped by hybridizing labeled DNA complementary to the RNA to blots of fragments of viral DNA. Labeled DNA complementary to Raji cell RNA was prepared by two methods. For most experiments, labeled DNA complementary to Raji cell RNA was prepared by hybridizing labeled viral DNA to RNA in solution and separating the DNA-RNA hybrid from denatured and reannealed DNA on cesium sulfate gradients, as described above (29, 41). After alkali denaturation of the DNA-RNA hybrid, labeled DNA selected for homology to the nuclear, polyadenylated, or polyribosomal RNA of Raji cells and induced Raji cells was hybridized to blots of separated EcoRI (Fig. 4) or BamHI (Fig. 5A) fragments of EBV DNA or to blots of separated cloned fragments of EBV DNA (Fig. 6). The DNA homologous to nuclear, non-polyadenylated, or polyadenylated RNA of Raji cells and the DNA homologous to nuclear or polyribosomal RNA of induced Raji cells hybridized to EcoRI fragments A, B, G, C, and F and, to a lesser extent, to EcoRI fragments Dhet and H (Fig. 4). In contrast, the DNA homologous to the polyribosomal RNA of Raji cells hybridized primarily to EcoRI fragment A (0.07 x 108 to 0.39 x 10' daltons) and, to a lesser extent, to EcoRI fragment B (0.56 x 108 to 0.75 x 10' daltons) (Fig. 4). On BamHI blots of viral DNA, the DNA homologous to the polyribosomal RNA of restringently infected Raji cells hybridized to BamHI fragments C, V, X, and H (map coordi-

653

nates, 0.05 x 108 to 0.29 x 10W daltons), to BamHI fragment K (0.63 x 108 to 0.66 x 108 daltons), and to BamHI fragment Jhet or Nhet (1.10 x 108 to 0.03 x 108 daltons) or to BamHI fragment I (0.90 x 108 to 1.02 x 108 daltons). The DNA homologous to the polyribosomal RNA of induced Raji cells hybridized to these same fragments and, to a lesser degree, to many other BamHI fragments, including BamHI fragments A, B, E, G, M, 0, P, and Z (Fig. 1 and 5). The intemal repeat (IR) in EBV DNA contains a single BamHI site which results in many copies of BamHI fragment V (6, 14) (Fig. 1). The right region of BamHI-C (0.100 x 108 to 0.115 x 108 daltons) contains the beginning of IR, and the left region of BamHI-X (0.241 x 108 to 0.246 x 108 daltons) contains the end of IR (5, 14). On blots of cloned BamHI fragments (Fig. 6), the DNA homologous to the polyribosomal RNA of restringently infected Raji cells hybridized more to the component of BamHI-C from 0.07 x 10" to 0.11 x 108 daltons than to the component from 0.05 to 108 to 0.07 x 10' daltons (EcoRI-J); it also hybridized to both the large and small BamHI/BglII components of IR or BamHI-V, to both HindIII components of BamHI-X (0.24 x 108 to 0.25 x 108 daltons) (data not shown), to BamHI-H (0.25 x 108 to 0.29 x 10" daltons), and to BamHI-K (0.63 x 108 to 0.66 108 daltons), but not to BamHI-I (Fig. 1 and 6). The DNA homologous to the polyribosomal RNA from induced Raji cells hybridized to the same DNA fragments and also hybridized to varying extents of BamHI fragments 0, L, E, B, A, and I (Fig. 1 and 6). In a control experiment in which labeled EBV DNA was hybridized to yeast RNA, treated with Si nuclease, and centrifuged twice in neutral cesium sulfate gradients, a small amount of labeled DNA was distributed throughout the gradients. The recovery of labeled DNA in the X

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FIG. 3. Hybridization of nuclear (A), polyadenylated (B), and polyribosomal (C) RNAs from restringently infected (O) or abortively infected (A) Raji cells to denatured 32P-labeled EBV (B95-8) DNA. The results for complexity and amounts referred to in the text and Table I are averages from this and two other similar experiments.

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region of the DNA-RNA hybrid was approximately 1% of the recovery obtained with DNA hybridized to Raji polyribosomal RNA. The labeled DNA did not hybridize to any fragments

blots of viral DNA. The identification of DNA encoding Raji RNA by using labeled viral DNA selected for homology to Raji RNA could enhance the relative contribution of DNAs having lower sequence complexity, such as IR and the terminal repeat, since these sequences were over-represented relative to the unique DNA sequences in on

TABLE 1. Amounts of RNAs in Raji cells and induced Raji cells % of Cells RNA DNABi %oRNAia

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(lanes R) or iododeoxyuridine-induced Raji cells (lanes RI) (A) or to 32P-labeled complementary DNA (cDNA) made by avian myeloblastosis virus reverse transcriptase from Raji polyadenylated (Poly A) polyribosomal RNA (B). The viral DNA used to prepare the blots was nearly completely digested with BamHI and appeared to be completely digested in pictures of the ethidium bromide-stained gel. However, since EBV (B95-8) DNA contains 10 tandem repeats of BamHI- V and the complementary DNAs have extensive homology to BamHI- V, a dimer of BamHI- V resulting from slightly incomplete BamHI digestion was

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Radiofluorograms of blots containing sepof EBV (B95-8) DNA which were hybridized to 32P-labeled EB VDNA (lane T.P.) or to 32P-labeled EBV DNA selected for homology to nuclear RNA, non-polyadenylated RNA [RNA (-PA)], polyadenylated RNA [(PA)RNA], or polyribosomal RNA of restringently infected (lanes R) or abortively infected (lanes RI) Raji cells. arated

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the labeled viral DNA originally hybridized to RNA and on the blots of fragments of viral DNA used for mapping. Nevertheless, even after equalization of the molar concentration of IR (BamHI-V) relative to other fragments by using blots of cloned BamHl fragments, DNA selected for homology to polyribosomal RNA hybridized intensively to BamHI-V (Fig. 6). To explore further the relative contributions Of IR and the unique viral DNA sequences to Raji polyribosomal RNA and to explain the differences between our results and the results obtained by another investigator with radioiodinated cytoplasmic RNA (48), we prepared DNA complementary to Raji polyribosomal polyadenylated RNA by using avian myeloblastosis virus reverse transcriptase. For these experi-

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ments polyribosomes were prepared by magnesium precipitation, and polyadenylated RNA was separated partially by one-passage chromatography on oligodeoxythymidylic acid cellulose as described above. On blots of viral DNA (Fig. 5B) the complementary DNA hybridized to BamHI fragments C, V, X, H, K, and Nhet or

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Jhet (map coordinates, 0.05 x 108 to 0.29 x 108, 0.63 x 10" to 0.66 x 108, and 1.10 x 108 to 0.03 x 108 daltons) (Fig. 1 and 5B). The hybridization to IR (BamHI-V) relative to the hybridization to BamHI-H was less with complementary DNA

than with viral DNA selected for homology to RNA, but no new unique sequence fragments were identified (Fig. 5A). On blots of cloned BamHI fragments (Fig. 7), the complementary DNA hybridized extensively to IR, to both HindIII components of BamHI-X, to the three largest HinfI components of BamHI-H, to BamHI-K, to the terminal BamHI component of EcoRI fragment Dhet-I, Jhet, and, a lesser extent, to BamHI-Bl (0.90 x 108 to 0.97 x 108 daltons). (DNA which maps at 0.93 x 10" to 0.96 x 10" daltons in BamHI-Bl is homologous to DNA at 0.25 x 108 to 0.29 x 108 daltons in BamHI-H [44].) A surprising finding was the marked increase in hybridization of this complementary DNA to the EcoRI-J component of BamHI-C, mapping at 0.05 x 108 to 0.07 x 108 daltons (Fig. 7A). This increase was disproportionate to the increase in hybridization to the other unique sequence DNAs, such as BamHIH (Fig. 7D) or BamHI-K (0.63 x 108 to 0.66 x 108 daltons in Fig. 7E). Therefore these data indicate that the marked increase in hybridization to EcoRI-J must have been due to a difference between the two procedures other than the sequence complexity effect. The Southern blot hybridizations in which labeled viral DNA selected for homology to Raji RNAs was used (Fig. 5A and 6) had less background than the hybridizations with DNA complementary to Raji RNA (Fig. 5B and 7). The formner procedure began with labeled viral DNA and involved selection of a fraction of the viral DNA which showed homology to Raji RNA. In the latter procedure or with radioiodination of RNA, more than 99.99% of the labeled nucleic acid (complementary DNA or RNA) was cellular and did not show specific homology to viral DNA. Hybridization of Raji RNA to BamHI-V (IR), to BamHI-H, and to EcoRI-J. The extent of homology and the relative amounts of the RNAs from IR and from the unique DNA sequences EcoRI-J and BamHI-H (0.05 x 10" to 0.07 x 108 and 0.25 x 108 to 0.29 x 108 daltons, respectively) were investigated further by hybridization of an excess of RNA to labeled DNA (Fig. 8 and 9).

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FIG. 7. DNAs of recombinant clones of EBV DNA digested with restriction endonucleases, separated on 0.6% agarose gels (lanes 1), blotted, and hybridized to 32P-labeled complementary DNA made from Raji cell polyadenylated polyribosomal RNA (lanes 2). The numbers which identify the EBV DNA fragments refer to the map coordinates (x108 daltons) on the map shown in Fig. 1.

Nuclear RNAs from Raji cells and from induced Raji cells hybridized to more than 60% of BamHI-V (Fig. 8). Polyribosomal RNAs from Raji cells and from induced Raji cells hybridized to more than 40% of BamHI-V, to more than 25% of BamHI-H, and to 12% of EcoRI-J (Fig. 8 and 9). Assuming that only one strand equivalent of DNA was informational and encoded cytoplasmic RNA, 80% of one strand of BamHIV, 50% of one strand of BamHI-H, and 24% of one strand of EcoRI-J encoded RNA. Identification of the abundant RNA encoded by EcoRI-J in restringently infected Raji cells. One hypothesis which could explain the enhanced hybridization of complementary DNA made from a magnesium-precipitated RNA template to EcoRI-J compared with complementary DNA prepared from polyribosomes purified by velocity centrifugation is that EcoRIJ encodes an abundant non-polyribosomal RNA which precipitates as a ribonucleoprotein complex (40). While these experiments were in progress, an abundant 160-base non-polyadenylated RNA encoded by EcoRI-J was discovered in the cytoplasm of IB-4 cells, a cell line which is growth transformed by EBV infection (53a). To determine whether a similar RNA was encoded by EcoRI-J in Raji cells, cytoplasmic RNAs from Raji and IB-4 cells were fractionated by oligodeoxythymidylic acid colunm chromatography, separated by size on agarose gels, blotted onto nitrocellulose filters, and hybridized to labeled EcoRI-J. As Fig. 10 shows, Raji cells contained an abundance of 160-base RNA encoded by EcoRI-J. From the relative intensities of hybridization of labeled EcoRI-J to the 160-base

RNAs in the non-polyadenylated RNA fractions of Raji cell cytoplasm and IB-4 cell cytoplasm (Fig. 10), the 160-base RNA was estimated to be fivefold more abundant in Raji cells than in IB4 cells; 18S and 28S RNAs were still discernable in the ethidium bromide-stained gel of the Raji cell polyadenylated RNAs, indicating that the polyadenylated RNAs were contaminated with non-polyadenylated RNA. Despite the incomplete separation of rRNA's from the polyadenylated RNAs, the level of the 160-base RNA in the polyadenylated RNA preparation was reduced fivefold by oligodeoxythymidylic acid cellulose chromatography (Fig. 10). Therefore, these data indicate that the 160-base RNA is not polyadenylated. Although two very-lowabundance polyadenylated RNAs have been detected with labeled EcoRI-J in IB-4 cells (53a), labeled EcoRI-J did not identify any larger RNAs in blots of polyadenylated or non-polyadenylated Raji cell RNA, nor did labeled EcoRIJ hybridize to any RNA in blots of polyadenylated or non-polyadenylated RNA extracted from non-EBV-infected lymphoblastoid cells (53a). DISCUSSION Our data are relevant to defining the relative levels of function of regions of the EBV genome in restringent and abortive infections and to defining elements in the regulation of expression of the EBV genome. An advantage of the Raji cell system is that different levels of virus expression can be studied in the same system. In the experiments described in this paper, Raji cells were induced with iododeoxyuridine, which resulted in detectable early antigen in 5 to 10% of

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the cells. More rapid and synchronous induction probably be achieved with phorbol esters or butyrate, inducers whose effects were discovered while our experiments were in progress (23, 25, 55). However, even with the limited induction obtained, changes in the mapping (Fig. 1) and processing of EBV-specified RNAs are apparent. At least three regions of the EBV genome (0.05 x 10W to 0.29 x 108, 0.63 x 108 to 0.66 x 108, and 1.10 x 10" to 0.03 x 108 daltons) (Fig. 1). encode polyribosomal RNA and presumably mRNA in Raji cells. These results are consistent with the less precise mapping data reported previously for the Burkitt tumor cell line, Namalwa (28, 41). Furthennore, our results are strikingly simrilar to the recently reported map of the DN.A encoding polyribosomal RNA in the IB-4 cell line, a restringently infected cell line established by growth transformation of normal lymphocytes with EBV (29, 53a). The similarity in the expression of the EBV genome in Raji and IB-4 cells suggests that the EBV genome can

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J. VIROL.

restringently infected Raji and IB-4 cells is that an abundant IB-4 polyribosomal RNA is encoded by DNA mapping at 0.54 x 108 to 0.59 x 108 daltons (Fig. 1). Polyribosomal RNA encoded by this region of EBV DNA is only detected in abortively infected Raji cells, suggesting that it encodes a polypeptide more abundant in abortive infections than in restringent infections. Polyribosomal RNA from abortively infected Raji cells is encoded by the three DNA regions which encode polyribosomal RNA in restringently infected cells and also by DNA mapping at 0.39 x 10" to 0.49 x 10", 0.51 x 10" to 0.59 x 10", 0.66 x 10" to 0.77 x 108, and 1.02 x 108 to 1.05 x 108 daltons (Fig. 1). Studies are in progress to map the DNAs of the early antigenassociated polypeptides (2, 25, 37) to defined sites in these regions. The defined DNAs should make it feasible to separate the components of the early antigen complex. Several results obtained in our experiments indicate that selective processing plays a role in the regulation of expression of EBV DNA in restringently infected Raji cells. First, the complexity of viral polyadenylated RNA is intermediate between the complexity of nuclear RNA and polyribosomal RNA, indicating that some RNAs selectively accumulate as polyadenylated RNA and that only some of the polyadenylated RNAs selectively accumulate on polyribosomes. Second, some of the nuclear RNA of restringently infected Raji cells is encoded by regions of the EBV genome (such as EcoRI fragments F and C) which are far removed from regions of adenylated TA (Poly A) IRNA were ind hybridhe EcoRI-J cell.,'s was estiwith brome markersiin separate

Raji cell cytoplasmic non-polyo Poly A) or polyadenylated RIA and IB-4 cytoplasmic non-polyadenylated run in parallel on agarose gels, blotted, a ized to 32P-labeled EcoRI-J. The size of tJ complementary RNA found in IB-4 mated to be 160 bases by comparison FIG. 10.

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the genome that encode polyribosomal RNA (Fig. 1 and 5). The long distances between EcoRI fragment F or C and segments of the EBV genome which encode polyribosomal RNA make it unlikely that RNAs from EcoRI fragments F and C are transcribed as parts of precursor molecules to polyribosomal RNA. Third, the observations in abortive infections that nuclear, polyadenylated, and polyribosomal RNAs are similar in complexity and that RNAs encoded by EcoRI fragments F and C accumulate on polyribosomes confirm that the observations made in restringently infected cells that polyribosomal RNA is less complex than nuclear RNA and that the RNAs encoded by EcoRI fragments F and C are not processed are not artifacts of the procedures used in the preparation of polyribosomal RNA. Therefore, these data suggest that promoters for transcription of some early antigen-associated RNAs may also function in restringently infected cells but that the RNAs are not processed. Identification of the sizes and map coordinates of specific nuclear RNAs encoded by these regions in restringent and abor-


VOL. 38, 1981

tive infections and of the polyribosomal RNAs

encoded by these regions in abortive infections could clarify this issue. ACKNOWLEDGMENTS T. Dambaugh, N. Raab-Traub, C. Beisel, M. Heller, D. Morse, M. Hawke, J. Dowling, and P. Morrison contributed materials or assisted in this work. John Taylor and Saul Silverstein contributed helpful advice regarding avian myeloblastosis virus reverse transcriptase and RNA blots. This work was supported by research grant MV32E from the American Cancer Society, and by Public Health Service grants CA 19264 and CA 17281 from the National Institutes of Health. W.K. and V.v.S. are trainees supported by Public Health Service Institutional National Research Service Awards AI 07099 and CA 09267 from the National Institutes of Health. E.K. is a faculty research awardee of the American Cancer Society.

ADDENDUM IN PROOF

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