JOURNAL OF VIROLOGY, Dec. 1992, p. 7239-7244

Vol. 66, No. 12

0022-538X/92/127239-06$02.00/0 Copyright © 1992, American Society for Microbiology

Characterization of Reticuloendotheliosis Virus-Transformed Avian T-Lymphoblastoid Cell Lines Infected with Marek's Disease Virus WILLIAM D. PRATT,1t ROBIN W. MORGAN,2 AND KAREL A. SCHAT'* Department of Avian and Aquatic Animal Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853-6401,1 and Department ofAnimal Science and Agricultural Biochemistry, University of Delaware, Newark, Delaware 197172 Received 19 June 1992/Accepted 21 August 1992 The expression of Marek's disease virus (MDV) transcripts and protein products was investigated in reticuloendotheliosis virus-transformed avian T-lymphoblastoid cell line RECC-CU91, which was superinfected with MDV. The presence of MDV in the superinfected cell line, renamed RECC-CU210, was demonstrated by Southern hybridization with 32P-labeled BamHI-H and -B fragments of the BamHI MDV DNA library. Examination of RECC-CU210 for the expression of MDV-specific RNA transcripts encoded by the internal repeat long (IRL), internal repeat short (IRS), and unique short (Us) regions of the MDV genome revealed two small transcripts of 0.6 and 0.7 kb. These transcripts were mapped to the IRL and IRS regions, respectively. In contrast, RECC-CU211, which was developed through transfection of CU210 with the BamHI-A fragment of MDV, expressed an additional nine transcripts from the IRL, IRs, and Us regions. CU211 but not CU210 also expressed a complex of polypeptides of 40, 38, and 24 kDa, identified by monoclonal antibodies as MDV-specific phosphoproteins. The 38-kDa phosphoprotein is likely to be pp38, an early viral protein that maps within the IRL region of the MDV genome. These findings suggest that genes located within the transfected BamHI-A fragment transactivated a number of genes located in the IRL region of the MDV genome.

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV), an oncogenic herpesvirus. Although MDV is classified as a gammaherpesvirus because of its biological characteristics (23), the structure of the MDV genome, as determined by electron microscopy (7) and restriction enzyme mapping (14), was found to be more like that of herpes simplex virus (HSV), an alphaherpesvirus. In addition, several MDV homologs of HSV genes have been identified and mapped to the unique short (Us) and inverted repeat short regions of the MDV genome (1, 5, 24). The discrepancies between the biological and structural characteristics of MDV are intriguing because certain features of MDV infection, such as latency and oncogenicity, are still unclear. In addition, these two facets of MD seem to be intimately associated, e.g., transformation appears to occur in latently infected T-lymphoblastoid cells. Currently, the mechanisms of transformation and latency are not known, yet they are fundamental to the understanding of MD pathogenesis. Recently, reticuloendotheliosis virus (REV)-transformed T-lymphoblastoid cell lines were developed (28) that could be superinfected with MDV and transfected with the BamHI-A fragment of MDV DNA (21). In this study, the REV-transformed cell line RECC-CU91 (CU91) was superinfected with MDV, resulting in a new cell line, RECCCU210 (CU210). CU210 was found not to express MDV antigens in the presence of 5-bromo-2-deoxyuridine (BUdR), suggesting that the MDV infection was latent in this cell line. However, when CU210 was subsequently transfected with pNL1, a selection plasmid containing the genes for ,-galactosidase and neomycin resistance, both under control of the *

simian virus 40 early promoter (28), and the BamHI-A fragment of MDV DNA, the new cell line, renamed RECCCU211 (CU211), expressed a complex of MDV-specific phosphoproteins (15, 29) at high levels. In contrast, transfection of CU210 with pNL1 alone resulted in a cell line, CU212, that did not express the phosphoproteins. The gene for one of these phosphoproteins, pp38, has recently been identified, sequenced, and localized to the BamHI-H fragment, to the left of the putative MDV origin of replication (9, 12). The promoter-enhancer region of pp38 overlaps that of a rightward BamHI-H 1.8-kb transcript that has been implicated in tumorigenicity (4). This region contains Oct-1, Spl, and CCAAT motifs in an arrangement similar to, although considerably less complex and multifarious than, the promoter-enhancer region for the ICP4 and ICP22 genes of HSV (32). The regulation of the pp38 and BamHI-H 1.8-kb genes and the functions of their protein products have yet to be established. In this study, the REV-transformed cell lines were characterized for MDV-specific transcripts encoded by the internal repeat long (IRL), internal repeat short (IRS), and Us regions of the MDV genome. We focused on these regions because previous studies reported finding viral transcripts only from these regions in MD tumor-derived cell lines (26, 30). In addition, expression of the MDV-specific phosphoproteins was examined.-

MATERIALS AND METHODS Cell lines. CU91 (29), CU210, CU211, and CU212 were developed previously (21) and maintained in LM-Hahn medium at 41°C (6). CU211 was selected from a number of biologically cloned cell lines that were all expressing pp38, as determined by an indirect immunofluorescent-antibody (IIFA) assay. CU211 was used for these studies because a higher percentage of cells were positive for pp38.

Corresponding author.

t Present address: Virology Division, USAMRIID, Fort Detrick,

MD 21702.

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PRATT ET AL.

TABLE 1. Characterization of REV-transformed cell lines Cell line

CU91 CU210 CU211

MDV DNA

detecteda + +

Parent

line

CU91 CU210

Transfected DNA fragment(s)' None None

pNL1, BamHI-A

% of cells expressing MDV-specific

polypeptidesc

+BUdR

-BUdR

0 0 22

0 0 16

CU212

+ 0 CU210 pNL1 0 a MDV DNA was detected by Southern hybridization with the BamHI-B and -H DNA fragments from the MDV DNA library (14) as probes (see Fig. 2). b DNA used in transfections: pNL1 DNA and BamHI-linearized BamHI-A fragment DNA from the MDV DNA library (14). c MDV-specific phosphorylated polypeptide expression in cell lines was subjectively determined by IIFA assay with H19.47, an MAb specific for pp38 (12). Cell lines were untreated (-BUdR) or treated with BUdR (20 pLg/ml) (+BUdR) for 48 h prior to examination for the presence of MDV-specific polypeptides.

Polyclonal chicken anti-MDV antibodies. Specific-pathogen-free chickens were infected with serotype 1 MDV, and sera were obtained at the time of tumor development. MAb and IIFA assays. The monoclonal antibodies (MAb) M21 and H19.47, both of which detect a complex of three MDV-specific phosphoproteins of 41, 39 or 38, and 24 kDa (pp38/41) (15, 29), were kindly provided by S. Kato (Osaka University, Osaka, Japan) and L. F. Lee (Avian Diseases and Oncology Laboratory, USDA, ARS, East Lansing, Mich.), respectively. IIFA assays were used to detect the MDV-specific phosphoproteins (27). Briefly, 105 acetonefixed cells were incubated with MAb for 15 min at 37°C, washed in 10 mM Tris-buffered saline (pH 8.7) for 10 min, and incubated with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin G (IgG) (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.). Cells were examined with a Leitz Dialux fluorescence microscope equipped with epi-illumination. Cell lines were incubated with or without the addition of 20 ,ug of BUdR (Sigma Chemical Company, St. Louis, Mo.) per ml for 48 h prior to examination for the presence of MDV-specific phosphoproteins (Table 1). Radioisotope labeling of viral proteins. Twenty million cells from the CU210, CU211, and CU212 cell lines were incubated for 4 h in methionine-free Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) containing 10% dialyzed fetal bovine serum. The cell lines were then incubated with 50 ,uCi of [35S]methionine (specific activity, 1,000 Ci/mol; Amersham Corp., Amersham, U.K.) for an additional 16 h at 41°C in a rocking waterbath. Labeled cells were washed three times in TNE buffer (100 mM NaCl, 50 mM Tris, 5 mM EDTA) and resuspended in 1 ml of lysis buffer (100 mM NaCl, 50 mM Tris, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 50 ,uM phenylmethylsulfonyl fluoride [Sigma Chemical Co.]). The cell suspensions were incubated for 1 h on ice and briefly inverted every 20 min to enhance lysis. The lysates were centrifuged for 10 min at 10,000 x g at 4°C, and the supernatant fluids were transferred to new tubes. The supernatant fluids were sequentially incubated on ice with normal rabbit sera diluted 1:5 in TNE and with 100 RI of 10% recombinant protein G (Life Technologies, Inc.) for 60 and 30 min, respectively. The lysates were centrifuged for 15 min, and the supernatant fluids were harvested and stored at -20°C until use.

RIP assays. Radioimmunoprecipitation (RIP) assays were performed as described previously (8). Briefly, aliquots of the preabsorbed supernatant fluids containing 2 x 107 dpm were incubated with 4 p. of MAb ascites fluid or 40 p.l of polyclonal chicken anti-MDV serum for 30 min on ice. The aliquots to which polyclonal chicken anti-MDV serum was added were further incubated for 30 min on ice with 20 R1 of 1:40 rabbit anti-chicken IgG (Cappel Laboratories, Cochranville, Pa.). Washed 10% recombinant protein G (100 p.l) was added, and the mixtures were incubated for 1 h with gentle mixing at 4°C. Following centrifugation for 15 s at 10,000 x g at 4°C, the supernatant fluid was removed, the pellets were resuspended in 1 ml of wash buffer (1 M NaCl, 0.1 M Tris-HCl, 0.1% Nonidet P-40 [pH 7.2]), and the mixtures were transferred to new Eppendorf microcentrifuge tubes. The mixtures were pelleted and washed twice in wash buffer,

and the supernatant fluids were discarded. The pellets were resuspended in 30 p1 of electrophoresis buffer (62.5 mM Tris-HCl [pH 6.8], 10% [vol/vol] glycerol, 2% [wt/vol] SDS, 0.05% [vol/vol] 2-3-mercaptoethanol, 0.00125% bromophenol blue), boiled for 10 min, quenched on ice, and centrifuged for 10 s at 10,000 x g. Supernatant fluids were stored at 4°C until use. Polyacrylamide gel electrophoresis. RIP samples were analyzed by the method of Laemmli (17). Samples were run in 12% polyacrylamide slab gels with a 4% polyacrylamide stacking gel on a Hoefer Vertical SE 400 gel apparatus (Hoefer Scientific Instruments, San Francisco, Calif.) at 150 V until the bromophenol blue dye marker ran to the bottom of the gel. Molecular weight standards (Life Technologies, Inc.) were used to estimate the sizes of the viral proteins. Gels were dried and exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, N.Y.) for 6 to 12 days. Southern hybridization. Total DNA was extracted from REV-transformed cell lines by standard procedures (26). DNA was digested overnight with the restriction enzyme BamHI as recommended by the manufacturer (Promega, Madison, Wis.). Digested fragments were separated by electrophoresis in 0.6% agarose and transferred to nylon membranes by Southern blotting. 32P-labeled probes were prepared from the BamHI-B fragment (located in the unique long [UL] sequence) and the BamHI-H fragment (located in the IRL flanking the UL and in the UL of the BamHI MDV DNA library [14]) with a random-primer kit (Multi-prime; Amersham Corp.) and [32P]dCTP (specific activity, 3,000 mCi/mmol; Amersham Corp.). Hybridization conditions were as described by Schat et al. (26). HindIII-digested lambda DNA (Sigma Chemical Co.) was used for size markers. Isolation of cellular RNA and Northern (RNA blot) hybridization. Total cellular RNA was isolated from lymphoblastoid cell lines with RNAzol B (BIOTECX Laboratories Inc., Houston, Tex.) and chloroform. All RNA preparations were stored in aliquots, precipitated with 1 volume of isopropanol, washed in 75% ethanol, and stored in 75% ethanol at -700C. Electrophoresis and Northern hybridizations were carried out essentially as described by Ausubel et al. (2). Briefly, precipitated RNA samples were washed in 75% ethanol and reconstituted in diethylpyrocarbonate-treated water, and the RNA concentrations were determined by measuring the A26. For each lane, 30 ,ug of RNA (5 to 11 pul by volume) was mixed with sample buffer (25 pu1 of formamide, 8.75 p.1 of 37% formaldehyde, and 5 p.1 of 10x MEA buffer [0.2 M morpholinopropanesulfonic acid (pH 7.0), 10 mM EDTA (pH 8.0), 80 mM sodium acetate]) and heated for 15 min at 55°C. Ten microliters of loading buffer (50% glycerol, 1 mM EDTA [pH

VOL. 66, 1992 TRL

REV-TRANSFORMED MDV-INFECTED AVIAN T-CELL LINES BamHI-

UL

IRL

IRS US TRs

A 1

2

B 3

X QLA,--'

A

1

4

f.## BamHI[H

7241

2

3 4

-1 8.3kb "

_

_ _ m

-11.4kb -5.5kb

- ICP4 - Hind I

PP38 FIG. 1. Schematic representation of the MDV genome. The BamHI linkage map of the region spanning the IRL, IRS, and Us is shown (14). The locations and orientation of the MDV ICP4 gene, the BamHI-B fragment, and the HindIII-3.1 fragment (1), which is located within the MDV ICP4 gene, are also shown. UL, unique long region; Us, unique short region; TRL and TRs, terminal repeats flanking UL and US, respectively; IRL and IRS, internal repeats flanking UL and Us, respectively.

8.0], 0.25% bromophenol blue, 0.25% xylene cyanole FF) was added and mixed, and the sample was loaded onto the gel. Electrophoresis was carried out in a horizontal 1.2% agarose-formaldehyde gel for 6 h at 100 V in lx MEA buffer. Chicken rRNAs of 28S (4.0 kb) and 18S (1.67 kb) were used as RNA size markers in the outside lanes. These lanes were removed, stained with ethidium bromide (0.5 ,ug/ml in 0.1 M ammonium acetate), and photographed. Gels were transferred to nylon membranes and hybridized for 18 h at 42°C in 5 ml of hybridization buffer (6 x SSC [1 x SSC is 0.015 M sodium citrate plus 0.15 M NaCl], 0.2% SDS, 5x Denhardt's solution (25), 50% formamide, 100 ,ug of denatured salmon sperm DNA per ml). Plasmids containing the BamHI-A, -H, -I2, or -L fragment of the BamHI MDV DNA library (Fig. 1) (14) were used as probes. In addition, the HindIII-3.1 fragment (Fig. 1), which is located within the BamHI-A fragment and within the MDV ICP4 gene (1), was also used as a probe. For each hybridization, the probe was added at a concentration of approximately 106 cpm/ml of hybridization buffer. After hybridization, the membranes were washed twice with 5x SSC-0.5% SDS at room temperature for 15 min, twice with lx SSC-0.5% SDS at 37°C for 15 min, and finally once 0.1x SSC-0.1% SDS at 65°C for 60 min. The filters were autoradiographed at -70°C with Kodak X-Omat AR film and intensifying screens. Films were exposed for 5 to 14 days. The efficiency of RNA transfer to the filters was evaluated by staining the filters in a solution of 0.5 M sodium acetate (pH 5.2) and 0.04% methylene blue (18).

FIG. 2. Southern blot analysis of CU91 (lanes 1), CU210 (lanes 2), CU211 (lanes 3), and CU212 (lanes 4) cell lines. Total DNA extracts were digested with BamHI, electrophoresed in a 0.6% agarose gel, and transferred to a nylon filter. MDV genome fragments were detected by autoradiography after hybridization to 32P-labeled BamHI-B (A) or BamHI-H (B) fragments from the cloned MDV DNA library (14).

postcloning). CU91, CU210, and CU212 were negative for pp38/41 expression even after incubation for 48 h in medium containing BUdR. The RIP assays gave similar results. CU211 expressed three MDV-specific polypeptides of 41, 39, and 24 kDa. pp38/41 was detected with both MAb M21 (data not shown) and MAb H19.47 (Fig. 4A). Only the 38-kDa MDV-specific phosphoprotein was precipitated from CU211 by polyclonal anti-MDV chicken serum (Fig. 4B). In addition, the polyclonal anti-MDV serum did not immunoprecipitate other MDV-specific proteins in CU211. The cell lines CU210 and CU212 did not express detectable levels of MDV-specific polypeptides with either the MAb or the polyclonal anti-MDV chicken serum. RNA transcripts in cell lines. The cell lines were examined for transcripts encoded by the BamHI-A, -I2, -H, and -L and HindIII-3.1 fragment regions of the MDV genome with probes prepared from the BamHI library and HindIII-3.1 (Table 2). MDV-specific transcripts were not detected in RNA extracted from CU91 (data not shown). (i) CU210 and CU212 cell lines. The CU210 and CU212 cell lines produced no detectable MDV-specific RNA from the

RESULTS

Establishment and characterization of cell lines. The three cell lines (CU210, CU211, and CU212) developed from the REV-transformed cell line CU91 were evaluated for MDV DNA by Southern blotting and for MDV-specific phosphoproteins by IIFA assays and RIP assays. CU210, CU211, and CU212 but not CU91 were positive for MDV DNA, as determined by Southern hybridization. The probe prepared from the BamHI-B fragment detected an 18.3-kb fragment (Fig. 2A). The BamHI-H probe detected the 11.4-kb band, corresponding to the BamHI-D fragment located in the terminal repeat flanking the UL and in the UL region and the 5.5-kb fragment corresponding to the BamHI-H fragment

(Fig. 2B).

In IIFA assays, CU211 was positive for pp38/41 with MAb (Fig. 3). The expression of pp38/41 was stable, allowing biological cloning of the cell line. The cloned cell line still expressed pp38/41 at 134 days posttransfection (= 9 days

FIG. 3. IIFA assay of CU211 cells stained with MAb H19.47, specific for the MDV phosphoprotein pp38 (12).

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J. VIROL.

PRATT ET AL.

A 1

41 K_ 39 K-

2

B 3

1

2

3

s-4 39K-

FIG. 4. RIP assay of cell lysates from CU210 and transfected CU210 cell lines. Cells (CU210, lanes 1; CU211, lanes 2; CU212, lanes 3) were labeled with [35S]methionine for 16 h and immunoprecipitated with protein A. (A) MAb H19.47 against MDV-specific phosphoproteins (29); (B) polyclonal chicken anti-MDV serum. The proteins were separated in 12% polyacrylamide gels. The locations of marker proteins are indicated by bars (105,700, 71,030, 44,230, and 27,770 Da).

BamHI-H or -I2 regions (Fig. 5B and C, lanes 1 and 2, respectively). No detectable MDV-specific transcripts from these cell lines were identified with the BamHI-A probe (Fig. 5A, lanes 1 and 2); however, the HindIII-3.1 probe hybridized weakly with a 0.7-kb transcript from both CU210 and CU212 (Fig. SE, lanes 1 and 2, respectively). In addition, CU210 and CU212 produced a moderately abundant 0.6-kb transcript that hybridized to the BamHI-L probe (Fig. 5D, lanes 1 and 2, respectively). (ii) CU211 cell line. Several additional MDV-specific transcripts were detected in RNA extracts from CU211. These were detected by hybridization with probes specific for the BamHI-A, BamHI-H, BamHI-12, and BamHI-L fragment regions. Transcripts from the BamHI-A and BamHI-12 regions were, in general, present in low quantities, resulting in weak signals. The estimated sizes of the MDV transcripts detected were 7.8, 5.9, 3.8, and 1.6 kb with the BamHI-A probe (Fig. 5A, lane 3), 3.2, 1.7, and 0.5 kb with the BamHI-H probe (Fig. 5B, lane 3), and 1.8 and 1.3 kb with the BamHI-12 probe (Fig. SC, lane 3). The pattern of transcription from the BamHI-L fragment region was similar to that in CU210 and CU212, with a single transcript of 0.6 kb (Fig. SD, lane 3). The HindIII-3.1 probe detected the 0.7-kb transcript in CU211 that was also observed in CU210 and CU212; in addition, it weakly detected a 3.8-kb transcript (Fig. SE, lane 3). TABLE 2. MDV-specific RNA transcripts in the avian lymphoblastoid cell lines CU91, CU210, CU211, and CU212r

MDV-specific RNA transcript(s) (kb)

Cell line

CU91 CU210 CU212 CU211

1

BamHI-H

BamHI-12

BamHI-L

None None None 3.2, 1.7, 0.5

None None None 1.8, 1.3

None 0.6 0.6 0.6

BamHI-A

None None

None 7.8,5.9, 3.6, 1.6

HindIII-3.1

None 0.7 0.7 3.6, 0.7

a The map locations of the BamHI-H through -A fragments of the BamHI library of the MDV genome (14) are given from the IRL to the US regions. The HindIII-3.1 fragment from the BamHI-A fragment is included.

2

3

-7.8 -5,9

-3.2

-3.6

-1.7

-1.6

-0.5

2

-1. .-

E

D

24K-

c

B

A 1 23

3

1 2

3

-3.8

-0.6

-0.7

FIG. 5. Expression of MDV transcripts in CU210, CU212, and CU211 cells. Total RNA extracts prepared from CU210 (lanes 1), CU212 (lanes 2), and CU211 (lanes 3) were electrophoresed in a 1.2% agarose gel and transferred to a nylon filter. MDV transcripts were detected by autoradiography after hybridization to 32P-labeled BamHI-A (A), BamHI-H (B), BamHI-I2 (C), or BamHI-L (D) fragment from the cloned MDV DNA library (14). The HindIII-3.1 fragment from the BamHI-A fragment was also used as a probe (E). The 28S (4.0 kb) and 18S (1.7 kb) rRNAs were used as size markers (arrowheads). RNA extracted from CU91 was hybridized with the same probes, but specific bands were not detected (data not shown). Sizes are shown in kilobases.

DISCUSSION

This article presents a possible in vitro model for viral latency during MDV infection of avian T lymphocytes. Our results characterized an REV-transformed cell line, CU210, which was superinfected with MDV and was MDV genome positive yet did not produce detectable MDV-specific polypeptides. Furthermore, this cell line did not produce any detectable transcripts from the BamHI-H or -I2 segment of the MDV genome, regions demonstrated to produce immediate-early (IE) transcripts (19, 26). CU210 also did not produce any detectable transcripts with the BamHI-A probe for the IRS and Us regions. However, a probe consisting of the HindIII-3.1 fragment from the IRS region of the MDV genome did detect an interesting 0.7-kb transcript from this region. Recently, Morgan (20), using this HindIII-3.1 fragment as a probe, also detected a 0.7-kb transcript in the MD tumor cell line MDCC-MSB-1 that was nonpolyadenylated and antisense to the MDV ICP4 gene. Our results are intriguing because several alphaherpesviruses are known to produce transcripts from the IRL or IRS region which are antisense to IE genes during latent infections. In varicella-zoster virus (11) and pseudorabies virus (22) but not in HSV, latency-associated transcripts from the Us region have been shown to be antisense to ICP4 homolog genes. In contrast, HSV has latency-associated transcripts which are antisense to the ICPO gene and are transcribed from the IRL region of the genome (reviewed in reference 13). Interestingly, the other transcript identified in CU210 was a 0.6-kb transcript that hybridized to the BamHI-L

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REV-TRANSFORMED MDV-INFECTED AVIAN T-CELL LINES

segment of the IRL region of the MDV genome. Several investigators have described numerous transcripts from the UL_, IRL, IRS, and Us regions in lytically MDV-infected cells and MD tumor cell lines (19, 26, 30) and in MDV-induced lymphoma cells (30). However, even the nonproducer MD tumor cell lines that showed the least MDV transcription, MDCC-HP1 (26) and MKT-1 (30), expressed at least four or more transcripts from the BamHI-A, -H, and -I2 regions of the MDV genome. The pattern of MDV gene transcription demonstrated in CU210 is novel and has not been described previously for MDV-infected cells. Recently, Cui et al. (12) and Chen et al. (9) independently identified, sequenced, and localized the gene encoding a 38-kDa MDV phosphoprotein, pp38. Treatment of MDVinfected lymphoblastoid cells with phosphonoacetic acid, an inhibitor of viral DNA synthesis, did not block the expression of pp38, indicating that pp38 may be an early viral protein (9). The 38-kDa protein in our results is most probably pp38, since both proteins are recognized by MAb H19.47 and both are coprecipitated with two other MDVspecific polypeptides, p40 and p24. Interestingly, our results showed that transfected MDV genes from the BamHI-A fragment (Us and IRS regions) of the cloned BamHI library of the MDV genome (14) may be able to transactivate transcription of genes located in the IRL region of the MDV genome in the latently MDV-infected REV-transformed lymphoblastoid cell line. This was demonstrated in CU211, which had been transfected with BamHI-A MDV DNA and subsequently expressed three MDV-specific polypeptides, of which at least one, pp38, appears to be encoded from the UL-IRL junctional region of the MDV genome (9, 12). Moreover, Northern hybridization analysis of CU211 confirmed that there was a 1.7-kb MDV transcript from the BamHI-H region, which approximates the size of the 1.8-kb transcript identified for pp38. In addition, two other MDV transcripts of 3.2 and 0.5 kb from the BamHI-H region were also identified. It is not surprising that the large, 23-kb BamHI-A region of the MDV genome would have the ability to affect the expression of the genes which are located within it. Herpesviruses are known to be promoter rich, and two herpesviral promoters, the cytomegalovirus IE enhancer-promoter and HSV thymidine kinase promoter, are commonly used as constitutively active promoters in eukaryotic expression vectors (3, 10). The finding that the Us and IRS regions may encode viral regulatory proteins that transactivate viral genes is also not unexpected. Recently, Anderson et al. (1) mapped the MDV homolog of the HSV IE protein ICP4 to the IRS region. In HSV, ICP4 has an essential function in the transactivation of numerous viral genes (reviewed in reference 31). It is not clear why the MDV genes in the transfected BamHI-A DNA appear to be expressed in CU211 cells while the MDV genes from this same location in the latent MDV genome in CU210 and CU212 cells are not. It is possible that

the BamHI-A DNA that was transfected into CU210 exists in a different state than the latent viral DNA, and three possibilities can be proposed. (i) A difference in the degree of methylation between the latently infected MDV genome and the transfected BamHI-A fragment DNA may contribute to the difference in MDV gene expression between CU210 and CU211. Methylation is thought to play a role in the restricted expression of MDV genes in MD tumor cell lines (16). In comparison, the viral DNA in productively infected cells is not methylated. Since the BamHI-A fragment was propagated in Escherichia coli, the transfected DNA would not be

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methylated, which may allow the genes within this fragment to be actively transcribed. (ii) The genomic location of the transfected MDV fragment may have enhanced the expression of MDV genes. In general, transfected DNA is thought to be randomly integrated into the cellular DNA of the transfected cell. However, since CU211 was biologically cloned as an MDV gene-expressing line, integration of the BamHI-A fragment into a chromosomal site that would induce the expression of MDV genes would have been preferentially selected. (iii) Other conceivable factors, such as recombination between the transfected MDV fragment and the latent MDV genome, cannot be ruled out. Further investigations are necessary to clearly understand our findings. ACKNOWLEDGMENTS This research was supported in part by Public Health Service grant ROICA0670-29 and grant 1-1460088 from BARD, the United States-Israel Binational Agricultural Research and Development Fund. We thank Priscilla O'Connell for excellent technical assistance. REFERENCES 1. Anderson, A. S., A. Francesconi, and R. W. Morgan. 1992. Complete nucleotide sequence of the Marek's disease virus ICP4 gene. Virology 189:657-667. 2. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1987. Preparation and analysis of RNA, p. 4.9.1-4.9.7. In Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 3. Boshart, M., F. Weber, G. Jahn, K. Dorsch-Hasler, B. Fleckenstein, and W. Schaffner. 1985. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41:521-503. 4. Bradley, G., G. Lancz, A. Tanaka, and M. Nonoyama. 1989. Structure of the MDV BamHI-H gene family: a gene potentially important for tumor induction, p. 69-75. In S. Kato, T. Horiuchi, T. Mikami, and K. Hirai (ed.), Proc. 3rd Int. Symp. Marek's Dis., Osaka 1988. 5. Buckmaster, A. E., S. D. Scott, M. J. Sanderson, M. E. G. Boursnell, N. L. J. Ross, and M. M. Binns. 1988. Gene sequence and mapping data for Marek's disease virus and herpesvirus of turkeys: implications for herpesvirus classification. J. Gen. Virol. 69:2033-2042. 6. Calnek, B. W., W. R. Shek, and K. A. Schat. 1981. Spontaneous and induced herpesvirus genome expression in Marek's disease virus tumor cell lines. Infect. Immun. 34:483-491. 7. Cebrian, J., C. Kaschka-Dierich, N. Berthelot, and P. SheldricL 1982. Inverted repeat nucleotide sequences in the genomes of Marek's disease virus and the herpesvirus of the turkey. Proc. Natl. Acad. Sci. USA 79:555-558. 8. Chandratilleke, D., P. O'Connell, and K. A. Schat. 1991. Characterization of proteins of chicken infectious anemia virus with monoclonal antibodies. Avian Dis. 35:854-862. 9. Chen, X., P. J. A. SondermeUer, and L. F. Velicer. 1992. Identification of a unique Marek's disease virus gene which encodes a 38-kilodalton phosphoprotein and is expressed in both lytically infected cells and latently infected lymphoblastoid tumor cells. J. Virol. 66:85-94. 10. Colbere-Garapin, F., F. Horodniceanu, P. Kourilsky, and A. C. Garapin. 1981. A new dominant hybrid selective marker for higher eukaryotic cells. J. Mol. Biol. 150:1-14. 11. Croen, K. D., J. M. Ostrove, L. J. Dragovic, and S. E. Straus. 1988. Patterns of gene expression and sites of latency in human nerve ganglia are different for varicella-zoster and herpes simplex viruses. Proc. Natl. Acad. Sci. USA 85:9773-9777. 12. Cui, Z., L. F. Lee, J. Liu, and H. Kung. 1991. Structural analysis and transcriptional mapping of the Marek's disease virus gene encoding pp38, an antigen associated with transformed cells. J. Virol. 65:5609-6515. 13. Feldman, L. T. 1991. The molecular biology of herpes simplex

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15.

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18. 19.

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