Cell. Vol. 61, 1315-1327,

June 29, 1990, Copyright

0 1990 by Cell Press

Expression of the BNLFI Oncogene of Epstein-Barr Virus in the Skin of Transgenic Mice Induces Hyperplasia and Aberrant Expression of Keratin 6 Joanna B. Wilson,’ Wendy Weinberg,t Robin Johnson,’ Stuart Yuspa,t and Arnold J. Levine’ * Department of Biology Princeton University F’rinceton, New Jersey 08544 t National Cancer Institute Eluilding 37, Room 3825 National Institutes of Health Bethesda, Maryland 20892

Summary The BNLF-1 gene of Epstein-Barr virus (EBV) encodes the latent membrane protein (LMP), one of the putative oncogene products of the virus. This gene has been expressed from two different enhancer-promoter constructs in transgenic mice, to determine its biological activity and possible contribution to oncogenesis. While transgenic mice expressing LMP in many tissues demonstrated poor viability, expression of LMP specifically in the epidermis induces a phenotype of hyperplastic dermatosis. Concomitant with the expression of LMP in this tissue (and in the esophagus) is an induction of the expression of a hyperproliferative keratin, K6, at aberrant locations within the epidermis. The epithelial hyperplastic phenotype caused by the LMP-encoding transgenes implies that the LMP plays a role in the acanthotic condition of the tongue epithelium in the human EBV- and HIV-associated syndrome oral hairy leukoplakia, as well as possibly predisposing the nasopharyngeal epithelium to carcinogenesis. Introduction Epstein-Barr virus (EBV) is one of the human herpesviruses and is the etiologic agent of infectious mononucleosis, a lymphoproliferative disorder. The virus is also causally associated with nasopharyngeal carcinoma, African Burkitt’s lymphoma, and other lymphoproliferative disorders occurring in immunosuppressed individuals (for review see Epstein and Achong, 1986). EBV has also been associated with oral hairy leukoplakia, a syndrome most commonly found in patients infected with the human immunodeficiency virus (HIV) and characterized by EBVpositive acanthotic lesions of the tongue epithelium (Greenspan et al., 1985). EBV enters a cell via the complement receptor type 2 (CR2) present on human B cells and certain epithelial cells (Fingeroth et al., 1984; Nemerow et al., 1985; Sixbey et al., 1987). EBV infection of human B lymphocytes in vitro yields, at high efficiency, proliferating immortal B lymphoblasts in which the viral genome is maintained as an autonomously replicating episome in a latent state. At least eight viral genes are consistently expressed in lym-

phoblastoid cell lines (LCLs) derived following EBV infection, all of which are candidates for involvement in the process of in vitro immortalization and also (by analogy) possibly in the disorders observed in vivo. As yet, EBV infection in epithelial cells in vitro has not been achieved. The BNLF-1 gene of EBV is one of the genes expressed in LCLs and encodes a latent membrane protein (LMP). The protein contains six hydrophobic segments predicted to be membrane-spanning domains, with both the aminoterminal and carboxy-terminal domains on the cytoplasmic side of the membrane (Liebowitz et al., 1986). Several lines of evidence suggest that LMP contributes to B cell immortalization. First, expression of the BNLF-1 gene in certain established rodent cell lines can induce the production of oncogenicfoci (Wang et al., 1985; Baichwal and Sugden 1988). Second, introduction of the gene into EBVnegative human LCLs induces the expression of three leukocyte cell adhesion molecules (LFA-1, LFA-3, and ICAM1) along with the IL-4-responsive surface antigen CD23/ FceRll and the transferrin receptor (Wang et al., 1988b), all of which may contribute to B cell growth regulation. Third, in human B cells and in rodent fibroblasts expressing LMP, the protein has been observed to colocalize with the intermediate filament protein vimentin (Liebowitz et al., 1987; Wang et al., 1988a) and cause an increase in the steady-state intracellular free calcium concentration (Wang et al., 1988b), both activities implicated in mediating signal transduction with relevance to cellular proliferation. Virtually all of these observations describing the phenotypes of the LMP product rest upon the action of this protein in established human B cell lines or established rodent fibroblasts in culture. The effect of LMP in normal cells in vivo has not been explored. For that reason, the experiments described here were initiated to follow the consequences of the expression of the BNLF-1 gene in epithelial cells (the other major cellular target of the virus) in transgenic mice and, if possible, to establish in vivo models of disease for both B cell and epithelial cell types. One advantage in the functional characterization of a product expressed in stratified epithelium is that a complete sequence of differential states can be examined simultaneously. At each differential stage as an epithelial cell progresses from the basal layer to the outer, terminally differentiated layer, different pairs of keratins are expressed, and these sets of keratins vary between epithelia (Fuchs and Green, 1980; Roop et al., 1983). Disruption of differentiation or hyperplastic events can be monitored by the alterations in keratin expression. For example, in the case of epidermal hyperplasia (and the human condition of psoriasis), the temporal and spatial regulation of keratin expression is disturbed (Knapp et al., 1987 [in the mouse]; Moll et al., 1982; Weiss et al., 1984; Stoler et al., 1988 [in humans]). In this study, the effects of expression of the BNLF-1 gene were investigated in transgenic mice. Expression of the LMP transcript and protein in certain epithelia has

Cdl 1316

LMP ENCODING TRANSGENES

PyLMP

EWLMP

Figure 1. Microinjected

Transgenes

The BarnHI-EcoAl fragment (top) of the plasmid pPyLMP and the Xbal-Clal fragment (bottom) of the plasmid pEuLMP were used for microinjection into mouse zygotes. EBV sequences are cross-hatched, the LMP coding region is in black, polyoma virus sequences are white, and the mouse immunoglobulin sequences are shaded with vertical striated lines. Defined enhancer sequences are represented by diamonds, and the polyoma origin of replication is indicated (ORI). Putative RNA start sites from mapped promoters are depicted with arrows, and the poly(A) addition signal of the BNLF-I gene is shown.

been analyzed with regard to the resultant phenotype and to alterations in keratin expression. The relevance of these data to human EBV-associated syndromes has been addressed. Results Production of Transgenic Mice Transgenic mice were produced as described by Brinster et al. (1985), using F2 generation zygotes from the two mouse strains C57BL16J and DBA/2J and injecting either PyLMP or EpLMP expression vectors (Figure 1). The two separate transcriptional control elements were used to ex-

Table 1. LMP Transgenic

Mouse Lines and Their Phenotypes Transcript

Location

2.5 kb

0.6 kb

PyLMP.5 (X-linked)

epidermis, tongue, muscle/bone

epidemis,

p~LMp.53~ (autosomal) PyLMP.9 PyLMP.54. 56, 57 PyLMP.6, 52

skin, (tongue)

tongue, (skin)

(skin) none none

tongue, skin tongue, skin (54) none

EPLMP.~~~

every tissue

specific tissues

EuLMP.43 (founder) EpLMP.40, 50, 51 EuLMP 39

NT

NT

NT (thvmus)

NT none

Mouse Line

press the BNLF-1 gene (encoding LMP) with the aim of enhancing expression either in epithelial cells or in E3 lymphocytes. For the former (PyLMP), the early region promoter and enhancer of polyoma virus (a mouse virus that itself causes many carcinomas) was chosen, and for the latter (EpLMP) the same promoter was maintained but the enhancer was replaced with the mouse immunoglobulin heavy chain (IgH) enhancer (see Experimental Procedures for details). The PyLMP vector has been shown to express the LMP protein and transform mouse cells in culture. DNA from tails of potentially transgenic mice was screened by Southern blot analysis using a BNLF-1 gene DNA probe. In the first series, of 48 offspring produced after injection of PyLMP sequences into zygotes, six males and six females were shown to carry the transgene (founder male numbers 9, 52, 53, 54, 55, and 57 and female numbers 5, 6, 17, 18, 56, and 58; see Table 1). Nine of these were successfully used to generate transgenic mouse lines, predominantly by backcrossing to the strain C57BL16J. The different lines were estimated to harbor between one and 15 copies of the transgene per cell. In the second series, of 38 offspring produced after injection of EuLMP sequences into zygotes, 15 pups were shown to carry the transgene. Of these, three were dead within 2 weeks of birth, leaving five males and seven females, from which eight viable mouse lines were generated. The different lines were estimated to harbor between one and 30 copies of the transgene (data not shown). Mice in both series, originating from four separate founders, PyLMP.5, PyLMP.53, EpLMP.33, and EuLMP.43, displayed a common phenotype in the skin (Table 1). However, in two of these instances, 33 and 53, the founder mice were mosaics of normal and transgenic cells and themselves displayed no phenotype while all positive progeny did so. Consistent with the idea that these ani-

Phenotypes

Alteration

rn K6 Expressron

epidermal hyperplasia, tongue, epithelial acanthosis, adult lesions (see text), coat color change, sex-linked lethality epidermal hyperplasia, coat color change coat color change normal normal

10x RNA increase in skin, 2.5x increase in tongue, abnormal protein location

PyLMP tongue

2.5x

RNA increase

RNA

in skin

none none none

EuLMP

Parentheses indicate very low expression. a Founders mosaic; only positive offspring

NT = not tested. displayed phenotype.

epidermal hyperplasia, lethality epidermal hyperplasia, lethality founders dead by 2 weeks normal

6x

RNA increase in skin, 31 x RNA increase in esophagus NT

NT none

EBV Oncogene 1317

in Transgenic

Mice

mals were mosaic, the estimated copy number of the transgene from DNA obtained from the offspring was higher than that in the founder mouse DNA and the frequency of offspring bearing the transgene was less than 50% in both cases (especially so for founder number 33, where only four out of 52 first generation [Gl] offspring were positive). Subsequent generations from line 53 maintained a constant transgene DNA copy number and gave rise to positive offspring at the expected Mendelian frequency of 50% with a constant phenotype, whereas lethality in the Gl-positive mice of line 33 prevented its further transmission. Female founder E~.LLMP.~~was very sickly and did not produce viable progeny. Consequently, of the phenotypically positive mice, only those from the PyLMP series were successfully maintained. Several Lines of Transgenic Mice Have the Phenotype of Epidermal Hyperplasia The founder mice PyLMP.5 and EpLMP.43 and the positive offspring from lines PyLMP5, PyLMP.53, and EkLMF?33 had in common a distinct phenotype of dermatosis giving a gross appearance of wrinkled, scurfy skin soon after birth (Figure 2, top). Histopathological examination (Figures 2A-2D) revealed hyperplastic thickening of the epidermis (an increased number of cell layers as compared with control animals) with a disorganization of the differentiating layers (least severe in mice from line 53). To various degrees (particularly in line 5) the phenotype increased in severity with age to a chronic dermatitis (Figures 2C and 2D) with increased acanthosis (thickening of the squamous epithelium), some hyperkeratosis, and chronic inflammatory infiltrates in the subcutaneous layers (also seen in the tongue of adult line 5 mice). Other notable differences between the transgenic positives and littermates that did not carry the transgene in these four lines included reduced size and altered fur and claw growth. The positives were runted at birth and continued to be smaller than negative siblings throughout their reduced life spans (most severe in line 5 and line 33 mice, which maintained on average half of the body weight of the negative siblings). In conjunction with the small size of the positive mice, there was a delay in some postnatal developmental features such as eye opening and fur growth compared with negative siblings. Subsequent fur growth continued to be affected in all four lines. Particularly notable in the PyLMP lines 5 and 53 and to a lesser extent in one other PyLMP line (number 9) was a gradual coat color change with age (from the characteristic black of the strain C57BL16J to ginger). Furthermore, the claws, another keratinous tissue, showed increased growth in the positives of the four lines in comparison with nontransgenic siblings. Lethality in the EwLMP Series Three founder mice in the EpLMP series (compared with none in the PyLMP series) were dead within 2 weeks of birth. Furthermore, mosaic founder female EpLMP.33, when crossed with C57BL16J males, produced only four progeny carrying the transgene out of a total of 52 offspring. All four positives displayed the phenotypes out-

lined above most severely and had a low viability (male 33.6 dead at 9 weeks; female 33.31 dead at 24 weeks; pup 33.47 dead at birth; and male 33.41 dying and sacrificed at 14 weeks) and none were able to breed (Table 1). Phenotypically positive founder female EpLMP.43 (detailed above) was fertile but unable to produce viable progeny. In the case of the phenotypically positive PyLMP lines, line 53 with an autosomal insertion of the transgene was viable, and line 5 with the transgene inserted on the X chromosome demonstrated sex-linked lethality (all positive males died soon after birth), probably owing to insertional mutagenesis. Together, these data indicate that the EpLMP transgene, unlike the PyLMP transgene, may be toxic. Late Onset of Adult Lesions in Line 5 Of all the phenotypically abnormal lines, line PyLMP.5 demonstrated the acanthotic thickening of the epidermis to the highest degree. On average the life span of these mice was short (the cause possibly complicated by an X-linked insertional mutation); however, the few long-lived adult females developed lesions that were never seen in negative siblings. To date, one adult female in this line developed a large, well-differentiated tumor of epithelial cell origin; hyperplastic but otherwise normal lymph nodes were also observed in this same mouse. A second mouse in this line developed chondrofibromas in the sternum. Various tissues (tongue, kidney, liver, and others) from a number of the mice displayed infiltrates of lymphocytes and histiocytes. Also, as noted above, line 5 mice as adults had a chronic dermatitis with notable inflammatory infiltrates in the subcutaneous tissue. Expression of the LMP Transcript Correlates with the Epidermal Hyperplastic Phenotype The pathologies in common to lines 5,53, and 33 and founder 43 suggested that there may be overlap in the specificity of expression of the transgene despite the use of different transcriptional enhancers to regulate the BNLF-1 gene. To investigate this possibility, the pattern of in vivo transcription from the transgene was determined. The predicted mRNA species that might be expressed from these transgenes (based upon known promoters) are diagramed in Figures 1 and 3 and include the following: first, the 2.5 kb LMP-encoding message; second, a 2.25 kb message driven from an internal promoter at late times during the EBV lytic cycle, encoding an N-terminally truncated LMP; and third, a 0.6 kb message driven from a second internal promoter (3’to the LMP coding region) at early times during the EBV lytic cycle (Hudson et al., 1965). With the possible message complexity in mind, the different RNA species were quantitated by Northern blot analysis. Total RNAs were purified from lo-day-old positive Gl pups derived from lines PyLMP.5 and PyLMP.53. The RNAs from each tissue were hybridized to the HindlllEcoRl fragment of the transgene (lacking the polyoma sequences, Figure 1). In line 5 (Figure 3A) the 2.5 kb LMPencoding transcript is expressed abundantly in the skin, with some expression in the tongue and a minor amount in muscle and bone. Similarly, this transcript is expressed

Cell 1318

Figure 2. Phenotype and Histopathology 5 and 53

of the Skin of PyLMP Lines

(Top) Photograph of Fday-old transgene-negative (left) and transgenepositive (right) siblings from the line PyLMP.53, before extensive fur growth, displaying the dermatosis skin phenotype and runting of the transgene-positive mice. (A-D) Skin sections from irday-old (A and B) and adult (C and D) line PyLMP.5 transgene-negative (A and C) and transgene-positive (B and D) siblings, stained with hematoxylin and eosin. The bar in (A) is 50 urn.

in the skin and tongue tissues from line 53 (Figure 36). This same RNA also hybridized with a first-exon probe, distinct from the 2.25 kb message (data not shown). The 2.25 kb.message was not detected but could have been masked by the larger 2.5 kb message owing to compaction in this area of the gel by the 18s ribosomal RNA. However a 0.6 kb message is highly abundant in the skin and the tongue from line 5 (Figure 3A) and tongue from line 53 (with a minor amount detected in the skin sample from line 53, Figure 38). Reprobing the same blots with various subfragments of the BNLF-1 gene revealed that this 0.6 kb message did not hybridize with the LMP coding sequences or the introns, but did with a fragment that exactly bounds

the predicted sequence of the EBV early lytic 0.6 kb message (data not shown). Unique to line 5, in every tissue analyzed except for the liver, two larger RNAs of approximately 4.5 and 10 kb were also detected hybridizing to the BNLF-1 gene probe. Thus, these two lines of mice (PyLMP.5 and 53) that display a distinctive phenotype of epidermal hyperplasia express the 2.5 kb LMP-encoding message and the 0.6 kb (early lytic) message in common in tissues of the skin and tongue. However, epidermal thickening is detected only in the tongues of line 5 mice (not 53) despite the abundance of the 0.6 kb message in both (Table 1). A comparison of expression from the tissues brain, skin,

EBV Oncogene 1319

in Transgenic

Mice

PyLMP . ... .... .. . ..... ....!

-

f-igure 3. Transgene-Specific

RNAs Expressed

+

2.5 hb

b

?‘ISLib 00kb

in PyLMP Lines 5 and 53

Twenty microgram samples of total RNA isolated from the indicated tissues from IO-day-old pups of line 5 (A) and line 53 (6) were Northern blotted and hybridized with BNLF-1 gene sequences. Included in (B) are 20 pg samples of total RNA from the human B ceil lines I84 (EBV-positive LCL), IRaji (EBV-positive Burkitt’s lymphoma line), and Louks (EBV-negative Burkitt’s lymphoma line). Below right: diagrammatic representation of possible transcripts from known promoters within the PyLMP sequences

and tongue from eight separate PyLMP lines is shown in Figure 4A. These tissues were derived from lo-day-old Gl pups from the phenotypically abnormal lines 5 and 53 and the phenotypically normal lines 6,52,54,56, and 57 (Table 1) and the above-mentioned line 9, which develops a mild phenotype of coat color change with age. Total RNAs obtained from a transgenic positive from each of the lines along with one negative sibling were blotted and probed with BNLF-1 gene sequences. The 2.5 kb LMP-encoding message can be readily detected in the skin only in lines 5 and 53, correlating well with the abnormal skin phenotype. The five phenotypically normal PyLMP lines presented here do not express detectable levels of the 2.5 kb LMP-encoding message (Figure 4A and Table 1). On very long exposure of the blot to film (not shown), a low level of this message is detected in the skin of line 9, correlating with the very mild phenotype which is accentuated when the line is bred to homozygosity for the transgene. The level of expression of the 2.5 kb LMP-encoding RNA was found to be 20-fold higher in line 5 than in line 53 and a further order of magnitude lower in line 9, when normalized for the amount of RNA loaded by rehybridizing the blot with the L32 ribosomal protein gene sequences. Six of the PyLMP lines analyzed (5, 9, 53, 54, 56, and 57) expressed the 0.6 kb early lytic message at various levels in tongue tissue (highest in lines 5 and 54), while lines 5,54, and 53 (in order of abundance) also expressed the transcript in the skin, but without any phenotypic correla-

tion (Table 1). Furthermore, the larger transcripts of 4.5 and 10 kb, expressed in most tissues in line 5, are specific to line 5 and consequently irrelevant to the skin phenotype that was shared by several independent transgenic mouse lines (Table 1). Therefore, in the PyLMP series of mice there is a direct correlation between the hyperplastic epidermal phenotype and the expression only of the 2.5 kb LMP-encoding transcript. Since mice in the EpLMP series (founder 43 and the offspring of mosaic founder 33) also displayed the hyperplastic dermatosis phenotype, the pattern of transcription from this transgene was also investigated. An extensive tissue expression analysis was performed on a Gl adult mouse of the line 33 (EpLMP.33.41, detailed above), which displayed the phenotype of epidermal hyperplasia in common with the PyLMP series but also showed poor viability. As a comparison, an age-matched transgene-positive mouse was taken from the phenotypically normal line EpLMP.39. Total RNAs derived from tissues of both mice were analyzed by Northern blot and hybridized with BNLF-1 sequences (Figures 48 and 4C). At this level of detection, on very long exposure of the blot to film, there is a low level of a 2.5 kb transcript observed in the thymus and spleen from line 39. By contrast, the 2.5 kb transcript was expressed in many tissues in line 33, being readily detectable in brain, muscle, nasopharyngeallpalate region, tongue, skin, esophagus, lymph nodes, and tail, and expressed at a lower overall level in all the other tissues

Cell 1320

Table 2. Comparison of the Transcript Levels from the ENLF-1 Gene and K6 Gene in EnLMP Mice Tissue

Ratio 2.5 kb Transcript=

Ratio 0.6 kb Transcripta

K6 33.41/ ControP

Nasopharyngeal region 10 35 NT Tongue 16 *1 450 Skin 25 90 8.0 Esophagus 33 630 31 a The level of the transcripts expressed has been normalized and presented as a ratio between tissues (Figure 6, arbitrarily assigning the intensity of the hybridization signal from the nasopharyngeal 2.5 kb transcript as 10). b The normalized ratio between the levels of K6 trancript observed from mouse 33.41 (Figure 86) and mouse 39.2 (Figure 8C). NT = not tested.

Figure 4. Transgene-Specific RNAs expressed PyLMP Lines and Two EnLMP Lines

in Eight

Different

Fifteen microgram samples of total RNA isolated from the indicated tissues were electrophoresed, Northern blotted, and hybridized with BNLF-1 sequences. (A) RNA was derived from the tissues brain (B), skin (S), and tongue (T) from IO-day-old positive pups from the PyLMP lines 5, 6, 9, 52, 53, 54, 56, and 57 and a transgene-negative control. RNAs from the human B cell lines Louks and I84 were included in the analysis. (Band C) RNAs were isolated from the indicated tissues from an adult mouse of line EnLMP.33 (8) and line EnLMP.39 (C). Included in (6) is RNA from the human B cell line 184.

tested. The 0.6 kb message was again expressed at high levels in the tongue and in the esophagus, at lower levels in skin, and in the tail and nasopharyngeal/palate region (the entire area was taken as one sample) (Figures 48 and 4C and Table 2). The correlation of expression of the 2.5 kb LMP-encoding message in the skin with the observed skin phenotype (seen in Figure 2) is confirmed by these

results (Table 1). Furthermore, the broad-range expression of this transcript may be relevant to the early lethality or toxicity associated with this transgene (EpLMP) versus PyLMP All the transgenic lines displaying the phenotype of epidermal hyperplasia express the LMP-encoding transcript in the skin (Table 1). To delineate this expression further, the skin was separated into dermal and epidermal layers. The skins from l-day-old Gl transgene-negative and transgene-positive siblings from line 5 were trypsin treated to separate the two layers. Total RNA was isolated from each layer (along with brain and tongue) and analyzed (as previously) by Northern blot hybridization. In skin, keratin expression is restricted to the epidermis while collagen expression is restricted to the dermis, and this fact was employed to test the specificity of the epidermis/ dermis separation technique. Using sequences unique to the mouse keratin 1, 10, and 14 (Kl, KlO, and K14) genes as probes, greater than 90% of these keratin messages segregated with the epidermal fractions (Figure 58). Using mouse collagen al(lll) and a2(l) sequences to reprobe the same blot, all of the detectable collagen messages segregated with the dermal fraction (Figure 5C), demonstrating the integrity of the separated fractions. Reprobing with BNLF-1 gene sequences revealed that greater than 80% of the LMP-encoding 2.5 kb message and all of the detectable 0.6 kb EBV early lytic message partitioned with the epidermal fraction, while the majority of the larger two messages specific to line 5 (4.5 and 10 kb) partitioned with the dermal fraction (Figure 5A). Thus, the correlation of epidermal phenotype with epidermal expression of the LMP-encoding transcript is strengthened by these results. The LMP Is Expressed in Hyperplastic Tissues The transcription of the LMP-encoding message tightly correlates with the phenotype of epithelial hyperplasia of the skin and tongue. To investigate the complete expression of the transgene into a protein product, tissue and cell extracts were immunoprecipitated with an affinity-purified polyclonal rabbit antiserum raised against the C-terminal half of LMP (Baichwal and Sugden, 1987). The immunoprecipitated proteins were separated by nonreducing SDS-

EBV Oncogene 13211

rn Transgenic

Mice

-28s

428s

-LMP

-Kl -KlO

,::;m 428s

-18s

418s

-18s

.KW

8

A Figure 5. EpidermallDermal-Specific

Transcripts

C

in PyLMP Line 5

Tnenty microgram samples of total RNA from the indicated tissues from l-day-old transgene-negative (-) and transgene-positive (+) pups from line 5 were Northern blotted and hybridized sequentially with BNLF-1 gene sequences (A), specific Kl, KlO, and K14 gene sequences (B), and collagen crl(ll) and a2(1) gene sequences (C).

PAGE and visualized by Western blotting employing the same anti-LMP serum as probe (Figure 6). Tissue extracts were prepared from the skins of 3-day-old pups of lines PyLMP.5 and PyLMP.53 and a transgene-negative control, from tongues of 3-day-old pups of line PyLMP.5, and also from skins of an adult of line PyLMP.5 and an age-matched negative control. In the same manner, an extract was prepared from the epithelial tumor that arose in one adult of the PyLMP.5 line (described above). As positive and negative controls for the LMP protein produced specifically ,from the PyLMP transgene, primary whole mouse embryo I(WME) cells were derived from lCday-old transgenepositive and transgene-negative embryos of mouse line PyLMP.5 and grown in tissue culture. Cell extracts were made from these cultures as well as from the 6 cell lines B-958 (EBV positive) and Louks (EBV negative). The LMP product of the described transgenes is predicted to be 16 amino acids smaller than the corresponding B-958 product, owing to the in-frame deletion of the third of five repeats in the gene (see Experimental Procedures). As such, by SDS-PAGE a slightly faster migrating band is seen to be expressed from the transgenic lines compared with B-958. LMP is detected in the skins of both PyLMP lines 5 and 53, in the tongues of line PyLMP.5, and in the epithelioid tumor that arose in line PyLMP.5 (Figure 6). This demonstrates that the transgene is fully expressed at the protein level, in a fashion predicted from the RNA expression data and interestingly in a “spontaneous” carcinoma derived from a line 5 mouse. A specific, larger band at approximately 120 kd is also detected in LMP-expressing tissues (with this and other LMP-reactive antisera; not shown) and may be a modified or complexed form of LMP

Keratin Expression The epidermal changes displayed in common by lines 5, 33, and 53 and founder 43 prompted an examination of the expression of epidermal cell markers. In particutar, the

z “! :

fi

co p.skin

&skin

AmABQ5355to5CAB

Figure 6. Expression

z q

of LMP in the PyLMP Lines 5 and 53

Protein extracts were prepared from 10’ Louks and B-956 cells, 2 x 10’ W M E cells derived from line 5 transgene-negative (A) and transgene-positive (6) embryos, dorsal skin from 3-day-old pups (pskin) from negative (-) and positive (5) siblings from line 5 (200 mg each), and 120 mg from a positive of line 53 (53) 50 mg of tongue tissue derived from 3.day-old positives of line 5 (5t), 400 mg of dorsal skin derived from a positive adult of line 5 (a.skin 5) and an age-matched negative control (askin -), and 370 mg of tumor tissue derived from a line 5 carcinoma (LX). Extracts were immunoprecipitated with a polyclonal rabbit antiserum to LMP and the products analyzed by SDSPAGE and Western blotting, employing the same antiserum and ‘*slprotein A-Sepharose as probe.

Cell 1322

Figure 7. Analysis of Keratin Expression

by Indirect lmmunofluorescence

Skin sections were taken from transgene-negative (A, C, and E) and transgene-positive (8, D, and F) pups of line 5, at 1 day (C and D) and 7 days of age (A, B. E, and F). (A) and (B) have been costained for Kl (green) and K14 (red) (yellow when overlapping). (C)-(F) have been costained for K6 (green) and K14 (red) (yellow when overlapping).

pattern of expression of the intermediate filament protein filaggrin and the cytokeratins 1, 6, 10, and 14 was examined. K14 is normally expressed in the basal layer of the epidermis (Fuchs and Green, 1980; Roop et al., 1983), while Kl, KlO, and filaggrin are markers of the differentiated suprabasal layers. K6 is normally expressed in the skin only in hair follicles and in the proliferating epidermis of wounds, but has been seen to be aberrantly expressed in the suprabasal epidermal layers in hyperplastic, neoplastic, and psoriatic skin (Knapp et al., 1987; Moll et al., 1982; Weis et al. 1984; Stoler et al., 1988).

Skin sections from transgene-negative and transgenepositive line PyLMP.5 siblings were probed using a biotinylated antiserum to mouse K14 (anti-mK14) visualized with streptavidin-Texas red. Antibodies directed against Kl, K6, or KlO or filaggrin were detected using a second antiserum conjugated to fluorescein isothiocyanate. Employing this double staining, the presence of K14 is visualized in red, the presence of Kl, K6, KlO, or filaggrin in green, and the presence of both K14 and one of the latter in yellow (Roop et al., 1987). Costaining with anti-mK14 and either anti-mK1 (Figures 7A and 7B), anti-filaggrin, or

EBV Oncogene 1323

rn Transgenrc

Mice

Figure 6. KG-Specific Transcripts Transgenic Lanes

Expressed

The Northern blots presented in Figure 4 gene sequence specific for K6. (A) Total RNAs from tissues of lo-day-old PyLMP lines (5, 6, 9. 52, 53, 54, 56. and 57) c:ontrol. The tissues analyzed include brain CT). (B and C) Total RNAs from adult tissues EuLMP.33 (B) and EuLMP.39 (C).

in PyLMP and EuLMP were rehybridized

with a

pups from eight different and a transgene-negative (B), skin (S), and tongue (as Indicated)

from line

anti-mK10 (data not shown) indicated that the normal epidermal basal layer expression of K14 was less restricted in the transgenic-positive mice (compared with negative siblings), being found to include suprabasal layers. Moreover, costaining with anti-mK14 and anti-mK6 (Figures 7C-7F) revealed completely aberrant expression of K6 in the epidermis, detectable even in l-day-old pups (Figures 7C and 7D). The normal hair follicle K6 expression was detected in the skin of the negative siblings (Figures 7C and 7E), while abnormal, suprabasal K6 expression was evident in the positives of line 5 (Figures 7D

and 7F). These results further attest to the association between epidermal hyperplasia and K6 expression. To investigate whether K6 expression was abnormal at the steady-state RNA level as well as at the protein level, the Northern blots presented in Figure 4 were rehybridized with a KG-specific gene sequence (Figure 8). K6 is normally constitutively expressed in the tongue epithelium, as demonstrated by all eight PyLMP lines and the negative control (Figure 8A) and the EpLMP lines 33 and 39 (Figures 88 and 8C). It also appears to be constitutively expressed in tissues of the adult tail (Figures 8B and 8C). The level of K6 expression in the hair follicles of normal skin is barely detectable under these conditions (readily detected on longer exposure of the blot to film); however, an overabundance of the steady-state level of K6 RNA in the skin of PyLMP line 5 is clearly evident (Figure 8A), paralleling the expression of the 2.5 kb LMP-encoding transcript. The variation in the amount of RNA loaded on the gel has been normalized against the ubiquitously expressed L32 ribosomal protein gene (by rehybridizing the blot with the L32 gene sequences). When normalized, line 5 intact skin RNA has a lo-fold higher steady-state level of K6 transcript than the negative control. Also, K6 expression in the tongue of line 5 and the skin of line 53 was seen to be 2.5-fold higher than in the negative control tongue and skin (respectively), which may be significant. K6 expression is also induced in line EpLMP.33 in the esophagus at least 31-fold and in the skin at least 8-fold (after normalization against the level of L32 expression; Table 2) in comparison with the line EpLMP.39 mouse, which does not express the LMP transcript (Figures 8B and 8C). Interestingly, when the LMP transcript is expressed in tissues of line 33 mice, which usually express no K6 at all (for example, brain and lymph nodes), K6 expression could not be detected, indicating that the induction of K6 by LMP is tissue specific. These results demonstrate a dramatic correlation between the expression of LMP in the epidermis of transgenie mice and a hyperplastic dermatosis phenotype and the abnormally induced expression of K6 in the same tissues. Discussion This report describes the effects of the expression of the BNLF-1 gene of EBV in transgenic mice. One transgene employed in this study (PyLMP) predominantly directs expression of LMP to the skin, giving rise to a phenotype of hyperplastic dermatosis with the induction and aberrant expression of K6 in epidermal cells. The second transgene used here (EuLMP) directs expression of LMP to a broad range of tissues, resulting in lethality as well as the hyperplastic dermatosis phenotype and K6 induction seen in common with the PyLMP transgene. Tissue Specificity of Expression Both the PyLMP and the E~ILMP transgenes have several promoters (indicated in Figure l), and the sequences involved in the specificity of expression are likely to be multiple and complex. Expression of the 2.5 kb transcript from

Cell 1324

PyLMP is at the highest level in the epidermis (in IO-dayold pups) and is probably affected by a combination of the intact polyoma virus promoter and enhancer, the interrupted BNLF-1 promoter, and internal gene promoters. Changing the polyoma early enhancer to the IgH enhancer appears to have expanded the repertoire of expression of the LMP message from tissues with a major component of stratified squamous epithelium (skin, tongue, esophagus, nasopharyngeallpalate region) to incorporate tissues with a major lymphoid component (lymph nodes, spleen, thymus) and, to some extent, many other tissues analyzed. The portion of the IgH enhancer used contains those sequences required for positive regulation of a heterologous gene, but only one of two possible negative regulatory elements proposed to restrict expression to I3 cells (Imler et al., 1987). Furthermore, the construct still contains the polyoma virus early and the LMP promoters, which undoubtedly contribute to the pattern of expression. Expression of the 0.6 kb RNA is specific mainly to tissues of the tongue, pharynx region, and esophagus and is probably driven by the EBV early lytic promoter 3’of the LMP coding region (Figure 1). The specificity of expression of this transcript is clearly parallel to the tissue pathology of EBV infection in vivo and the putative sites of viral lytic replication. Consequently, viral replication may be tightly cell-type restricted at the level of lytic promoter activity (and not solely at the level of viral entry into a cell via a receptor). This particular early lytic promoter, in the absence of other viral gene products, is not detectably functional in tissues of lymphoid composition. The expression of this 0.6 kb RNA in these transgenic mice did not correlate with any phenotype described in this study (Table 1). Expression of LMP and Phenotypic Correlation in Transgenic Mice Of the eight PyLMP transgenic lines analyzed, the two lines with readily detectable levels of the 2.5 kb LMPencoding message (by Northern blotting) and the protein product (by immunoprecipitation and Western blotting) were the only two displaying the hyperproliferative disorder of the skin described herein. This correlation is further supported by the detection of the LMP message in the skin of an EuLMP line also displaying the hyperplastic dermatosis phenotype. The detection of this message (unlike the 0.6 kb transcript) directly correlates with the epidermal hyperplastic phenotype, demonstrating that epithelial expression of LMP in vivo induces hyperplasia. Furthermore, in a line where expression of the transgene is highest (PyLMP.5) tumorigenic lesions appear later in the life of the mice, and one of these lesions (a tumor of epithelial origin) was demonstrated to be expressing LMP Although tumorigenic lesions were seen as rare events in this one line, similar lessons were never observed in the negative siblings, and it is possible that the LMP-induced hyperplasia predisposed these tissues to tumorigenicity. The near-ubiquitous expression of the 2.5 kb transcript in tissues of the nonviable EuLMP transgenic line 33 as compared with a second line (39) without detectable expression or lethality (Table 1) lends further evidence to the

toxicity of LMP (Hammerschmidt et al., 1989) as may the death of three founders in this series by 2 weeks of age. Keratin Expression The changes in keratin expression in the epidermis in line 5 are similar to those observed previously with the conditions of hyperplasia, neoplasia, and psoriasis (Knapp et al., 1987; Moll et al., 1982; Weiss et al., 1984; Stoler et al., 1988). These include expression of K14 (normally restricted to the basal layer) in the basal and suprabasal layers, concomitant with Kl and KlO suprabasal expression. More dramatic is the aberrant expression of K6 (normally restricted to hair follicles and wounds in the skin) in the suprabasal layers. This pattern of expression has been shown at the protein level in line 5, as well as by a substantial increase (lo- to 30-fold induction) in the steadystate level of K6 RNA in lines 5 and 33. The direct correlation between the expression of LMP and the epidermal hyperplastic phenotype extends to the induction of K6. That is, in the PyLMP lines where the LMP transcript is expressed predominantly in the skin with less in the tongue, K6 is quantitatively induced. Conversely, despite the expression of the 0.6 kb message in the tongue of many PyLMP lines (e.g., line 54; Figure 4 and Table l), there is no concomitant increase in K6 expression in the absence of the 2.5 kb transcript. mRNA expression analysis of the EpLMP line 33 (Figures 4 and 8 and Table 2) demonstrates that the LMP-mediated K6 induction is tissue specific. The 2.5 kb transcript is expressed in many tissues, but K6 mRNA induction is limited to certain tissues in which a major component is stratified epithelium. Whether LMP induces other keratins (e.g., K16, or keratins expressed in the claws, since elongated claw growth was observed), or indeed other intermediate filaments in different tissues, is under investigation. The data presented here do not support a simple model of direct gene trans-activation, since there are tissues (line 33) in which the LMP transcript is expressed but K6 is not induced. The increase in K6 mRNA may reflect the association between the expression of cell type-specific intermediate filaments and the growth state of the cell. There are many possibilities as to how LMP expression could achieve this phenotype. It has been suggested that LMP may have an ion channel-like structure. Epithelial cells are exquisitely sensitive to ion concentrations, particularly Ca2+ (Hennings et al., 1980) such that perturbation of the cellular ion concentrations by LMP could disrupt the normal differential state (or, conversely, growth state) of the cell, which dictates the keratin types to be expressed. Since LMP has been shown to induce the expression of and interact with vimentin in human 6 cells and rodent fibroblasts (Liebowitz et al., 1986; Wang et al., 1988a), LMP may also directly interact with one or more of the keratins at the protein level, interfering in the intermediate filament network. Pertinence to Human Disease If these findings indeed reflect the action of LMP in human cells, LMP could be responsible for the acanthosis and parakeratosis seen in the human EBV- and HIV-associ-

EBV Oncogene 1325

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Mice

ated condition, oral hairy leukoplakia (Greenspan et al., 1985). The entire LMP promoter sequences (disrupted in the transgene construct) may normally direct expression to the epithelium of the tongue, esophagus, and pharynx (as does the 0.6 kb RNA promoter in this context). Acanthotic thickening was indeed observed in the tongue epithelia of adult mice of line PyLMP.5 in which LMP was expressed. Many tissues of adult line PyLMP.5 mice showed chronic inflammatory infiltrates. As LMP has been shown to induce the cell surface expression of certain adhesion molecules (Wang et al., 1988b), this infiltrating lymphocytic activity may be mediated by LMl? If so, it suggests an intriguing mechanism whereby infectious epithelial EBV can increase its access to circulating lymphocytes. The expression of LMP alone, in the absence of any cellular alterations, other viral antigens, or chemical age+ does not immediately induce the onset of carcinomas (at least through its expression in the epidermis of the mouse). However, by generating a hyperplastic condition in association with the abnormal expression of the K6 gene in the epidermis, LMP may predispose these cells to oncogenesis. Indeed, a spontaneous carcinoma derived from an LMP transgenic mouse was found to be expressing the protein. Whether long-term expression of LMP, or its expression in conjunction with other factors, increases the incidence of cancer is under study. Experimental

Procedures

Plasmid Constructions for the Transgenes The BNLF-1 gene encoding LMP of the 895-8 strain of EBV was isolated from the cosmid pCB281 (Dambaugh et al., 1980). Our isolate of thts cosmid stock harbors a 48 bp in-frame deletion relative to the published 895-8 sequence, removing the third of the five repeats present In the C-terminal half of the LMP coding region. Different EBV isolates vary in their number of these repeats (Hennessy et al., 1984), and the isolate used here is active in the assay of transformation of Rat-l cells using the plasmid PyLMP (see below). The sequences used were from the Mlul site at 169,572 (converted to a Hindlll site for this construction) to the BamHl site at 166,616. These sequences contain the upstream TATA-like (TACATAA) element, the cap site for the message, the entire LMP transcribed sequences (including the two introns and polyadenylabon signal), and 329 bp 3’ of the poly(A) signal. Polyoma virus sequences encompassing the enhancer, the viral origin of replication, and the early promoter (TATA) and cap site were isolated from the plasmid p43.2b.67 (polyoma virus A2 strain) (Tyndall et al., 1981) from the HamHI site at position 4632 to the BstXl site at 170 (the latter converted to a Hindlll site for this construction). The EBV and polyoma virus sequences were linked through the introduced Hindlll sites to produce the plasmid pPyLMP (Figure l), with approximately 15 bp from the polyoma virus early cap site to the start of the EBV sequences. For the plasmid pEpLMP, the BamHI-EcoRI fragment from the mouse Igti locus, incorporating the intronic enhancer (Banerji et al., 1983), was isolated and used to replace the polyoma enhancer from the plasmid PyLMP An EcoRl linker was introduced at the Pvull site at polyoma virus position 5265 in PyLMP (3’ of the enhancer, 5’of the origin of replication) and the BamHI-EcoRI fragment replaced with the @I enhancer fragment. The 3816 bp BamHI-EcoRI fragment from pPyLMP (including 30 bp of pBR sequences at the 3’end of the gene) and the 4020 bp Xbal-Clal fragment from pEbLMP (including 6 bp of pBR sequences at the S’end of the gene) were isolated and used for microinjection into mouse zygotes (Figure 1). Transgenic Mice Transgenic C57BL16J x DBAlPJ mice were generated according to the

procedures established by Brinster et al. (1985). Where possible, founder mice were backcrossed to the inbred strain C57BU6J to generate lines. In the case of line 5, the outbred mouse strain CD1 was used. Production of Transgenic WYE Cells Embryos of 14 to 17 days’ gestation were isolated and finely minced (retaining a portion for transgene DNA analysis). The slurries were trypsinized for 40 min at 37% and strained through gauze. The cells were washed and then plated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Subsequently, cells predominantly fibroblastic in nature were maintained in culture. Genomic DNA Analysis Mouse tail DNA was isolated by digestion of the intact tissue in 1% SDS, 150 m M NaCI, 10 m M Tris-HCI (pH 7.5), 100 m M EDTA (pH 8.0) with 0.5 mglml proteinase K at 55% overnight. The genomic DNA was purified by one extraction with buffered phenol followed by one extraction with buffered phenol-chloroform, then precipitated by the addition of ammonium acetate to 1.25 M and an equal volume of cold ethanol. Direct centrifugation at 8.000 x g for 2 min selects against the precipitation of RNA. The DNA was digested according to the enzyme manufacturer’s recommendations, agqrose gel electrophoresed, and denatured for Southern blot analysis. The DNA was transferred to Biodine nylon membrane (ICN) electrophoretically in a Hoefer electroblotting apparatus for 3 hr at 30 V in TAE buffer and then UV cross-linked to the membranes. For slot blot analysis, undigested denatured DNAs were transferred to Biodine nylon under vacuum. Blots were hybridized with DNA fragments labeled with [&*P]dCTP by the random primer polymerization method (Feinberg and Vogelstein, 1983, 1984). Hybridizations were carried out as described by Church and Gilbert (1984). Filters were washed at high stringency, in final washes of 0.1x SSC, 0.1% SDS at 68OC, prior to autoradiography. Transgene copy number estimates were obtained from densitometric analysis of slot-blotted DNA derived from positive offspring of the founder transgenlc mice. RNA Isolation and Analysis Total RNA was isolated from frozen tissues essentially as described by Chomczynski and Sacchi (1987). Approximately 15-20 pg of total RNA was denatured in formamide at 68°C and electrophoresed through denaturing formaldehyde gels for Northern transfer. Included in many of the gels was a radiolabeled RNA ladder (system from BRL) electrophoresed with 15 to 20 vg (as appropriate to the gel) of transgene-negative RNA to allow for nbosomal RNA distortton. Gels were equilibrated in TAE buffer, and the RNA was electrotransferred to Biodine nylon for 2.5 hr at 35 V In TAE buffer. UV cross-linking, probe hybridizations, and washes were carried out under the same conditions as for Southern blot analysis (above). Northern blots were stripped of probe sequences in 75% formamide, 0.1x SSC, 0.1% SDS at 65% for 30 mln, followed by two 30 min washes in 0.1x SSC. 0.1% SDS at 65% prior to rehybrldlzation. EpidermallDermal Layer Separation For this procedure mouse pups less than 24 hr old were used (before extensive fur growth through the epithelium). By a single dorsal incision, the entire skin was removed and floated dermis side down for 5 hr at 4% on a 5 ml solution of buffered trypsin (5 m M KCI, 0.44 m M KH2P04, 140 m M NaCI, 4 m M NaHC03. 5.5 m M o-glucose. 0.34 m M Na2HP04, with 0.375% bovine pancreatic trypsin type Ill) containing 45 m M ribonucleoside-vanadyl complex. The skins were then placed epidermis side down on plastic and the dermis was removed and snapfrozen separately from the intact epidermis, which was also snapfrozen in liquid NZ. DNA Probes and Normalization of Loaded RNA Short gene fragments isolated from cDNA clones, consisting primarily of the 3’ noncoding regions, were used as probes for specific keratin sequences. For K6 a 290 bp fragment was used (Greenhalgh and Yuspa, 1988), and for Kl, KlO, and K14, 400 bp, 300 bp, and 450 bp fragments, respectively, were used (Roop et al., 1983). A 500 bp Xbal fragment from the plasmid pMCS1 (Liau et al., 1985) was used as a collagen al(lll)-specific probe, and an 850 bp Xhol frag-

Cell 1326

ment from the plasmid PAZ1002 (Schmidt et al., 1984) was used as a collagen a2(1)-specific probe. The probe used to analyze transcripts derrved from the BNLF-1 gene was the Hindlll-EcoRI fragment isolated from pPyLMP (Figure 1). For normalizing the amount of total RNA loaded in each lane of a gel, Northern blots were stripped and reprobed with a sequence specific to the ribosomal protein L32 transcript, a 1.1 kb Hindlll fragment from the plasmid p4ASac1.6 (Dudov and Perry, 1984). Densitometric values were obtained and used as reciprocals for the readings taken for other transcripts from the same sample. lmmunoprecipitation and Western Analysis Protein extracts were prepared from snap-frozen tissue samples and frozen ceil pellets in the same manner. The samples were homogenized in a nonionic detergent buffer (50 m M Tris [pH 81, 150 m M NaCI, 5 m M EDTA [pH 81, 0.05% NP-40, 0.5% Triton X-100) with the protease inhibitors aprotinin to 2% and phenylmethylsulfonyl fluoride to 100 mM. The samples were centrifuged at 10,000 x g for 20 min at 5°C and the pellets resuspended in Ripa buffer (50 m M Tris [pti 81, 150 m M NaCI, 1% Triton X-100, 1% deoxycholic acid, 0.5% SDS) with the same protease inhrbitors as above. The samples were centrifuged again and the supernatants collected for immunoprecipitation. The SDS was diluted to 0.1% by addition of the nonionic detergent buffer, and the samples were precleared with protein A-Sepharose (Sigma). An affimty-purified polyclonal rabbit antiserum (Baichwal and Sugden, 1987) was used at 3% sample volume and precipitated wtth an equal volume of protein A-Sepharose. Precipitates were washed twice with nontonic detergent buffer (without Triton X-100) and once with Tris-buffered saline. Proteins were separated by nonreducing SDS-PAGE (10%) and then electrophoretically transferred to Immobilon-P membrane (Millipore). Membranes were blocked for nonspecific binding with PBS containing 5% nonfat milk, and specific proterns hybrrdized wrth the antiserum at 1.5% total volume. Bound antibodres were detected with 1251-labeled protern A. Employing this technique, a pro portion of the LMP is extracted with the noniomc detergsnt step as predicted (Eaichwal and Sugden, 1987); in Figure 6, the remainmg Ripa-extracted material is presented. Indirect lmmunofluorescence of Skin Sections Monospecific anti-keratin antibodies were used to localrze keratrn expression rn a double-label Indirect immunofluorescence stalnmg reaction. Sections of Carnoy’s frxed skins were Incubated with affinitypurified serum from two species injected with synthetic peptides corresponding to the cDNA clones for mouse Kl, K6, KlO (rabbit, diluted 1 500) or K14 (guinea pig, diluted 1:2000) (Roop et al., 1984, 1985). After 18 hr at room temperature, sections were washed and incubated for 30 min wrth preimmune rabbit serum (1:200) and biotinylated goat antiguinea pig IgG (1:lOO; Vector Laboratory, Burlingame, CA). Antibody blnding was visualized following a final 30 min Incubation with strep tavidm-Texas red (1:400; Bethesda Research Laboratories, Gaithersburg, MD) and fluorescein isothiocyanate-conjugated swine antirabbrt IgG (1:40; Dako Corp., Santa Barbara, CA). All reagents were diluted in PBS contarnmg 12% BSA. Sections were examined and photographed under a Nikon Labophot microscope containing an epifluorescence attachment (HMXHBO 100 W lamphouse), using G and B2E filter blocks and Ektachrome 400 ASA film. Acknowledgments We would like to thank Dr. B. Sugden for the krnd grft of an afflmtypurified polyclonal antiserum to LMP and Dr. Denis Roop for the plasmids containing 3’ sequences of the mouse Kl, K6, KIO, and K14 genes. We acknowledge the expert histopathalogical analyses by Dr. Alan Rabson. We are also grateful to Dr. Stephen Pilder for his critical readrng of the manuscript. This work was supported by National Cancer Institute grants CA49271-02 and CA3875%04. J. 8. W. was a recipient of a Merck and Co. postdoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received March 13, 1990; revised April 20, 1990.

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Wang, D., Liebowitz, D., and Kieff, E. (1985). An EBV membrane proteini expressed in immortalized lymphocytes transforms established rodent cells. Cell 43, 831-840. Wang, D.. Liebowitz, D.. Wang, F., Gregory, C., Rickinson, A., Larson, R., Springer, T., and Kieff, E. (1988a). Epstein-Barr virus latent infectrorl membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity. J. Virol. 62, 4173-4184. Wang, D., Liebowitz. D., and Kieff, E. (1988b). The truncated form of the Epstein-Barr virus latent-infection membrane protein expressed in virus replication does not transform rodent fibroblasts. J. Virol. 62, 2337-2346. Wellss, R. A., Eichner, R., and Sun, T.-T (1984). Monoclonal antibody analysis of keratin expression in epidermal diseases: a 48- and 56kdalton keratin as molecular markers for hyperproliferative keratinocytes J. Cell Biol. 98, 1397-1406.