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[Cancer Research 64, 2167-2174, March 15, 2004]
© 2004 American Association for Cancer Research

Immunology

Epstein-Barr Virus Infection of Human Natural Killer Cell Lines and Peripheral Blood Natural Killer Cells

Yasushi Isobe1, Koichi Sugimoto1, Lixin Yang2, Kenji Tamayose1, Motoki Egashira1, Takako Kaneko1, Kenzo Takada2 and Kazuo Oshimi1

1 Division of Hematology, Department of Medicine, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, and 2 Department of Tumor Virology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although considerable part of natural killer (NK) cell neoplasms possess EBV genome, there has been no direct evidence that EBV infects human NK cells in vitro. In this study, we demonstrated EBV entry into NK cells using a recombinant EBV, which contains enhanced green fluorescent protein (EGFP) gene in its genome (EGFP-EBV). After 48 h of exposure to EGFP-EBV, we detected EGFP signals in ~30% of NK-92 and NKL cells and >40% of peripheral blood NK cells from three healthy volunteers. Reverse transcription-PCR analysis of various EBV-associated genes confirmed EBV infection. In situ hybridization for EBERs and BHLFs showed that latent and lytic infections coexisted at the early phase of EBV infection in two NK cell lines. Although BHLF-positive cells in the early lytic phase were round-shaped, EBER-positive cells in latent EBV infection tended to show a bizarre shape. Flow cytometric analysis of EGFP-EBV-exposed NK cell lines showed that most of EBV-infected cells entered early apoptosis after 72 h of EBV exposure, which explains the difficulties to establish EBV-carrying NK clones. Flow cytometry and reverse transcription-PCR analysis indicated that two NK cell lines may fuse with EBV using HLA class II after binding to the virus through a distinct molecule from CD21. We established two EBV-carrying NKL clones showing latency types I and II, both of which are recognized in EBV-associated NK cell neoplasms. Because EBV-infected NKL cells showed only type I latency during the early phase of infection, the temporal profile of latent gene expression is similar to that of T cells. We first report in vitro EBV infection of human NK cells and establishment of EBV-carrying NK clones, which should contribute to elucidate the role of EBV in the development of NK cell neoplasms.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EBV is a human herpesvirus carried by the majority of human population. Primary EBV infection causes infectious mononucleosis. Persistent EBV infection is associated with various lymphoid and epithelial malignancies, including Burkitt lymphoma, Hodgkin lymphoma, peripheral T-cell lymphoma, and nasopharyngeal carcinoma (1 , 2) . Recent reports suggest that the EBV infection is closely associated with mature natural killer (NK) cell malignancies (3, 4, 5, 6) .

Lymphoproliferative disorders of mature NK cells consist of nasal-type NK cell lymphoma, aggressive NK cell leukemia/lymphoma, and chronic NK lymphocytosis (7, 8, 9, 10, 11) . Nasal-type NK cell lymphoma frequently arises in eastern Asia and Central and South America. Some cases of lethal midline granuloma correspond to nasal-type NK cell lymphoma. Necrosis and angiodestructive infiltration of tumor cells are histological features of the disease (6 , 7) . The patients present coagulopathy and multiorgan failure at the advanced stage, and the prognosis is very poor (8) . Aggressive NK cell leukemia/lymphoma is a fatal disease, which is characterized by high fever, lymphadenopathy, hepatosplenomegaly, and atypical granular lymphocytes proliferation in the peripheral blood (7 , 9) . In contrast, most cases of chronic NK lymphocytosis are asymptomatic and require no treatment (10 , 11) .

EBV entry into target cells usually results in lytic or latent infection (2 , 12) . In the lytic infection, the viral structural genes are all transcribed, which leads to virion production and the resultant cell lysis of host cells. The latent infection is predominantly nonproductive and characterized by expression of six EBV-determined nuclear antigens genes (EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA-LP), three latent membrane proteins genes (LMP1, 2A, and 2B), BamHI A rightward transcripts, and two abundant EBV-encoded nonpolyadenylated RNAs (EBER1 and EBER2; Refs. 2 , 12 ). Lymphoblastoid cell lines transformed by EBV in vitro demonstrate the full pattern of gene expression, which is defined as type III latency. In contrast, the latent gene expression is more restricted in EBV-associated malignancies (1 , 2) . Burkitt lymphoma expresses EBERs, EBNA1, and BamHI A rightward transcripts, which corresponds to type I latency. In Hodgkin lymphoma, peripheral T-cell lymphoma, and nasopharyngeal carcinoma, a range of the latent gene expression is extended to LMPs. The pattern corresponds to type II latency. In EBV-associated NK cell malignancies, there have shown that the gene expression pattern corresponds to type I or II latency (13 , 14) .

Many studies have shown that the neoplastic NK cells possess monoclonal EBV genome, which indicates that latent EBV infection occurs before the clonal expansion (3, 4, 5) . In almost all of the cases of nasal-type NK cell lymphomas, tumor cells contain EBV (6 , 7) . This suggests that EBV should play an important role in the pathogenesis of the disease. EBV is also frequently detectable in the Japanese cases of aggressive NK cell leukemia/lymphoma (11 , 14) . Chronic NK lymphocytosis with hypersensitivity to mosquito bites is known as a distinct entity related to chronic active EBV infection (15) . The proliferating NK cells are infected with EBV. Such cases occasionally take a progressive course and consequently lead to aggressive NK cell leukemia/lymphoma (15 , 16) . However, the manner of EBV infection has never been elucidated in human NK cells.

EBV entry into B cells requires multiple interactions between viral and cellular molecules. Binding of the major viral capsid glycoprotein, gp350/220, to CD21 mediates the initial EBV attachment to B cells (17 , 18) . Viral internalization into B cells depends on an additional interaction between gp85-gp25-gp42 viral glycoprotein complex and HLA class II molecules (18, 19, 20, 21) . Recent studies, however, suggest that CD21-independent mechanism may exist for EBV infection, especially in epithelial cells (21, 22, 23, 24, 25) .

In this study, we detected the EBV entry into human NK cells and cell lines using the recombinant EBV (rEBV) that contains enhanced green fluorescent protein (EGFP) gene in its genome (25 , 26) . This method clearly shows EBV infection even before the establishment of persistent infection.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Virus.
In this study, we used two types of rEBV: neor-EBV and EGFP-EBV. In neor-EBV, the neomycin resistance gene (NEOr) is inserted at the site of BXLF1, which encodes EBV thymidine kinase. EGFP-EBV contains NEOr plus reporter EGFP gene at the same site to visualize EBV infection in living cells. Details of rEBV generation are described previously (25 , 27) .

Cell Lines.
As the target cells, we used NK-92 and NKL, which were derived from leukemic cells of each aggressive NK cell leukemia/lymphoma patient (28 , 29) . Both NK cell lines are grown in Iscove’s modified Dulbecco’s medium (Invitrogen, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Intergen, Purchase, NY), 2 mM glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), 100 µg/ml streptomycin (Invitrogen). Human interleukin-2 is essential for maintenance of both NK cell lines (100 units/ml, provided by Shionogi, Osaka, Japan). Akata, a Burkitt lymphoma-derived cell line, infected with neor-EBV or EGFP-EBV, was used as rEBV-producing cells (30 , 31) . Akata was maintained in RPMI 1640 (Sigma, St. Louis, MO) with the same supplements and 700 µg/ml G418 (Invitrogen). Raji (EBV-positive, CD21-positive) and U937 (EBV-negative, CD21-negative) were used as positive and negative controls, respectively. They were supplied from Japanese Cancer Research Resources Bank.

Purification of Peripheral Blood NK (PB-NK) Cells.
PB-NK cells were isolated from heparinized venous blood obtained from three healthy volunteers. NK cell-rich mononuclear cells were isolated by centrifugation in Ficoll-Hypaque gradient using RosetteSep NK cell cocktail (StemCell Technologies, Vancouver, British Columbia, Canada). PB-NK cells were purified by negative depletion with OKT3 (anti-CD3; Ortho Diagnostic Systems, Raritan, NJ), BL1a (anti-CD5; Coulter, Miami, FL), and B1 (anti-CD20; Coulter) monoclonal antibodies (mAbs). Positively labeled cells were removed with Dynabeads goat anti-pan mouse IgG (Dynal, Oslo, Norway). Trypan blue-dye exclusion procedure showed that >99% of the purified cells were viable. Evaluation by CYTRON ABSOLUTE flow cytometer (Ortho Diagnostic Systems) confirmed that the preparation contained >98% of NK cells (CD3-CD16+ or CD3-CD56+ cells).

Virus Production and Infection Procedure.
We obtained infectious rEBV from Akata cells by surface immunoglobulin cross-linking as described previously (31) . Each rEBV concentrate was resuspended into 1.5 x 107 particles/µl. The concentrate (250 µl) was added to 250 µl of the cell suspension (2.5 x 106 cells) in a polypropylene tube. Cells were incubated with rEBV for 2 h at 37°C and resuspended (2 x 105/ml) in culture medium and incubated. After 48 h of incubation, the cells were harvested and analyzed. The other cells were reseeded into 96-well plates at 102/well in culture medium containing 700 µg/ml G418 (Invitrogen).

Reverse Transcription-PCR (RT-PCR) Analysis.
Briefly, cDNA synthesis was performed from 800 ng of total RNA and 100 pmol of random hexamer (Takara, Otsu, Japan) using Superscript II reverse transcriptase (Invitrogen) in a volume of 20 µl. For PCR amplification, 1 µl of cDNA was added to 49 µl of PCR reaction mixture containing 1.25 units of TaqDNA polymerase (Takara). To analyze EBV-associated gene transcripts, we performed nested PCR analysis. Details of the sequences of primers and PCR conditions are given in Table 1Citation .


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  		Table 1 Oligonucleotides used for reverse transcription-PCR analysis

 
Propidium Iodide (PI) Rejection and Annexin V Binding Assays.
We evaluated cell death process of rEBV-exposed NK cell lines by PI and annexin V binding assays using Annexin V-Biotin Kit (Coulter). Each cell suspension (4 x 106/ml) of both NK cell lines was exposed to EGFP-EBV as described above. The cells were collected and washed with CaCl2-containing binding buffer (Coulter). Each cell suspension (2 x 104/ml) was mixed with 2.5 µg/ml PI or biotinylated annexin V (Coulter) for 10 min at 4°C. Cells mixed with PI were subsequently washed, fixed with 2% paraformaldehyde/PBS for 20 min at room temperature, and analyzed. Cells mixed with biotinylated annexin V were washed, resuspended in 100 µl of binding buffer, and incubated with ECD-labeled streptavidin for 30 min at room temperature. They were washed, fixed with 2% paraformaldehyde/PBS, and analyzed by flow cytometry.

Detection of EGFP by Fluorescence Microscopy and Flow Cytometry.
rEBV-exposed and control cells were collected, washed once, and resuspended at the concentration of 5 x 105/ml in 35-mm Glass Bottom Microwell Dishes (Mat Tek, Ashland, MA). The green fluorescence was analyzed using Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). Part of these cells were directly fixed with 0.4% paraformaldehyde/PBS and analyzed by flow cytometry.

RNA in Situ Hybridization.
Cells were collected, washed twice with 1x PBS, and concentrated. Cell preparations on slide glasses were fixed with 4% paraformaldehyde/PBS at room temperature for 20 min. After treating them with proteinase K (DAKO, Glastrup, Denmark), we used FITC-labeled peptide nucleic acid probes for EBERs (EBER1 and EBER2) and BamHI H fragments, lower strand frames (BHLFs) (BHLF1 and BHLF2; DAKO), and evaluate the expression using PNA ISH Detection kit (DAKO) according to the manufacture’s instructions.

Cell Surface and Intracytoplasmic Expression Analysis by Flow Cytometry.
One x 105 cells were labeled with various mouse mAbs described below, followed by the addition of phycoerythrin-conjugated goat antimouse mAb (Coulter). Fc{gamma} receptors on the cell surfaces were blocked with human immunoglobulin (provided by Mitsubishi Pharma, Osaka, Japan). The labeled cells were analyzed by flow cytometry. We used OKB7 (IgG2a; Ortho Diagnostic Systems) and anti-CR2 (IgG2a; Becton Dickinson, San Jose, CA) for CD21 and I3 (IgG2a; Coulter) for {alpha} and ß complex of HLA class II. For detection of surface markers, we used anti-CD2 (Coulter), anti-CD3 (DAKO), B1 (anti-CD20; Coulter), and anti-CD94 (Coulter). Freshly isolated NK cells were stained with FITC-conjugated Leu-4 (anti-CD3 mAb; Becton Dickinson), phycoerythrin-conjugated NKH1 (anti-CD56 mAb; Coulter), and phycoerythrin-conjugated 3G8 (anti-CD16 mAb; Coulter). We also analyzed intracytoplasmic LMP1 expression with CS1–4 (mouse anti-LMP1 mAb; DAKO), using paraformaldehyde-saponin procedure as described elsewhere (37) .

Western Blot Analysis.
Cell lysates were prepared as previously described (38) . Equal amount of total cellular protein (50 to 100 µg) prepared from each sample was separated on a discontinuous SDS-10% polyacrylamide gel, and blotted onto nitrocellulose membrane (Bio-Rad, Hercules, CA). Expression of EBNA2, LMP1, ZEBRA, and early antigen-diffuse (EA-D) was examined by using PE2, CS1-4, BZ1 (DAKO), EA-D-p52/50 (Chemi-Con, Temecula, CA), respectively. Antibody signals were enhanced using Envision polymer (DAKO) and detected with ECL Western blot detection system (Amersham International plc, Buckinghamshire, United Kingdom) according to the manufacturer’s protocol.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infection of Human NK Cell Lines by rEBV.
We examined EBV entry into NK-92 and NKL cells using EGFP-EBV, which contains the EGFP gene in its genome. Approximately one-third of NK-92 and NKL cells were EGFP positive on confocal laser microscopy after 48 h of exposure to EGFP-EBV. Flow cytometric analysis confirmed that ~30% of both NK cell lines were positive for EGFP signal (Fig. 1)Citation . Because nonexposed and neor-EBV-exposed cells were negative for EGFP signal, appearance of EGFP-positive cells after EGFP-EBV exposure verified EBV infection of both NK cell lines.



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  		Fig. 1. Enhanced green fluorescent protein (EGFP) expression in NK-92 and NKL cells infected by EGFP-EBV. NK-92 (A) and NKL (B) were incubated for an additional 46 h after 2 h of exposure to medium, neor-EBV or EGFP-EBV. Confocal laser microscopy (top column) and flow cytometry (middle and bottom columns) detected EGFP signal only in EGFP-EBV-exposed NK-92 and NKL cells.

 
Detection of EBV-Associated Gene Transcripts in rEBV-Exposed NK Cell Lines.
To confirm EBV infection, we performed RT-PCR analysis of viral transcripts after 48 h of exposure to rEBV. In nonexposed NK-92 and NKL cells, we detected no EBV-associated gene transcripts, indicating that both NK cell lines are free from EBV infection. After 48 h of exposure to neor-EBV or EGFP-EBV, we detected EBER1 in NK-92 and NKL cells (Fig. 2A)Citation . These data indicated that neor-EBV, as well as EGFP-EBV, infected both NK cell lines. Among six viral transcripts evaluated, we detected transcripts of Qp-initiated EBNA1, BZLF1, and BALF2 but none of EBNA2, LMP1 or LMP2A in rEBV-exposed NK-92 and NKL cells (Fig. 2B)Citation . BZLF1, which encodes lytic transactivator ZEBRA, and BALF2, which encodes a major DNA binding protein, are expressed in the lytic infection. These findings raised the possibility that latent and lytic infection coexisted in both rEBV-exposed NK cell lines. In situ hybridization showed that EBERs and BHLFs were transcribed in neor-EBV-exposed NKL cells (Fig. 3)Citation . EBERs are known as markers for latent infection, whereas BHLFs, which encode proteins belonging to the subgroup of EA-D, are transcribed in the lytic infection. Highly deformed bizarre cells tended to be positive for EBERs’ signals (Fig. 3)Citation . On the contrary, BHLFs were mainly transcribed in round-shaped cells (Fig. 3)Citation . These data suggest that latent and lytic phase population coexisted in neor-EBV-exposed NKL cells. We observed essentially the same tendency in neor-EBV-exposed NK-92 cells (data not shown). These results confirmed that rEBV infects NK-92 and NKL cells.



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  		Fig. 2. Reverse transcription-PCR analysis of EBV-associated gene expression in recombinant EBV-infected natural killer (NK) cell lines. A, EBER1 expression in neor-EBV- or EGFP-EBV-exposed NK-92 and NKL cells. B, nested reverse transcription-PCR analysis detected EBNA1, BZLF1, and BALF2 but not EBNA2, LMP1, or LMP2A transcripts in recombinant EBV-exposed NK-92 and NKL cells. As a positive control, we used Raji in for EBNA1, EBNA2, LMP1, and LMP2A and Akata treated with 20 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) for 8 h for BZLF1 and BALF2.

 


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  		Fig. 3. RNA in situ hybridization of EBERs and BHLFs in neor-EBV-exposed NKL and established clone 1. We used Raji and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated Akata as positive controls for EBERs and BHLFs, respectively. After 48 h of exposure to neor-EBV, we detected both EBERs and BHLFs signals in NKL cells. However, only EBERs’ signal was detected in clone 1.

 
EGFP-Positive NK Cell Lines Entered Apoptotic Process.
As shown in Fig. 1Citation , we found that a considerable part of EGFP-positive NK cell lines showed a bizarre shape. Cell deformation was also observed in neor-EBV-exposed NK cell lines. We, then, examined whether EBV infection causes cell death in the host cells. EGFP-EBV-exposed NK cell lines were evaluated by PI rejection and annexin V-binding assays. After 48 h of exposure, no annexin V or PI signal was detected in NK-92 and NKL cells. By 72 h, however, most of EGFP-positive NK cell lines were stained with annexin V, although they remained negative for PI signal (Fig. 4)Citation . These data indicate that most of NK-92 and NKL cells entered early phase of apoptosis by 72 h after EBV-EBV infection.



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  		Fig. 4. Propidium iodide (PI) rejection and annexin V binding assays in EGFP-EBV-exposed natural killer (NK) cell lines. NK-92 (top two columns) and NKL (bottom two columns) were evaluated for cell death after exposure to EGFP-EBV. At 48 h, no PI or annexin V signal was detected in both NK cell lines. At 72 h, EGFP-positive NK-92 and NKL cells were positive for annexin V but not for PI. As a positive control, we treated both NK cell lines treated with 1 mM etoposide for 12 h as a positive control.

 
NK Cell Lines Lacked CD21 but Expressed HLA Class II.
EBV is known to engage CD21 in the initial attachment to target cells. We examined CD21 expression on the surface of NK-92 and NKL cells by indirect immunofluorescence and flow cytometry. We used two mAbs, OKB7 and anti-CR2, which recognizes the short consensus repeat 1-2 and the short consensus repeat 3-4 of the CD21 molecule, respectively. As shown in Fig. 5ACitation , both antibodies failed to react with NK-92 or NKL cells, indicating the lack of the CD21 expression in both NK cell lines. RT-PCR analysis, which amplifies the short consensus repeat 2-3 site of the CD21 molecule, confirmed that these cell lines lack the CD21 transcripts (Fig. 5B)Citation . We next examined the expression of HLA class II, which acts as a coreceptor in EBV infection. Flow cytometric analysis clearly detected {alpha} and ß chain complex of HLA class II on the cell surface of NK-92 and NKL (Fig. 5A)Citation . These findings show that both NK cell lines lack the main EBV receptor CD21 but express HLA class II ß chain, which bind to gp85-gp25-gp42 complex.



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  		Fig. 5. HLA class II but not CD21 expression levels in NK-92 and NKL cells. A, flow cytometric analysis detected HLA class II but not CD21 in NK92 and NKL cells (solid histogram). Open histogram shows the result obtained with a control monoclonal antibody. B, reverse transcription-PCR analysis detected no CD21 transcripts in NK-92 and NKL cells. In A and B, Raji and U937 were used as positive and negative controls, respectively.

 
Infection of PB-NK Cells by rEBV.
Because NK-92 and NKL were proved to be target cells of EBV, we next evaluated whether this event was applicable to the normal PB-NK cells. We purified PB-NK cells from normal peripheral blood mononuclear cells using immunomagnet beads by the negative-selection method. Flow cytometric analysis showed that ~98% of the isolated cells were CD3-CD16+ or CD3-CD56+ (Fig. 6A)Citation . The purified cells were exposed to rEBV in the same way as the two NK cell lines. At least 98% of the medium-exposed PB-NK population remained CD3-CD16+ or CD3-CD56+ for 48 h (Fig. 6A)Citation . Confocal microscopy detected EGFP signal in EGFP-EBV-exposed PB-NK cells after 48 h of incubation. Flow cytometric analysis confirmed that >45% of PB-NK cells were EGFP positive (Fig. 6B)Citation . EGFP signal was detected in ~74 and 90% of EGFP-EBV-exposed PB-NK cells from two other volunteers (Fig. 6C)Citation . In comparison to the nonexposed PB-NK cells, rEBV-exposed ones became larger in size. Giant cell formation was obvious in addition to cell deformation in PB-NK cells after rEBV infection.



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  		Fig. 6. EBV infects normal peripheral blood natural killer (PB-NK) cells. PB-NK cells purified from normal donors were exposed to medium, neor-EBV, or EGFP-EBV. A, approximately 98% of the purified cells were CD3-CD16+ or CD3-CD56+ even after 48 h of incubation. B, confocal laser microscopy (top column) and flow cytometry (bottom column) detected enhanced green fluorescent protein (EGFP) signal only in EGFP-EBV-exposed PB-NK cells. C, more than 70% of PB-NK cells from other two volunteers were also positive for EGFP signal.

 
Establishment of EBV-Carrying NKL Cells.
After G418 selection, we obtained two G418-resistant clones containing EBV from neor-EBV-exposed NKL cells. Although we tried to establish EBV-carrying NK-92 and PB-NK cells, most of rEBV-exposed cells died within 1 week after the start of G418 selection. In comparison to original cells, the two established NKL clones tended to have nuclear deformation such as indented nuclei or micronuclei (Fig. 7A)Citation . Surface marker expressions such as CD2, CD3, CD20, and CD94 were essentially the same among original and two obtained NKL clones (Fig. 7B)Citation .



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  		Fig. 7. Characterization of two EBV-carrying NKL clones. Two G418-resistent clones were obtained from neor-EBV-exposed NKL cells. A, Wright-Giemsa staining of two established clones (clone 1 and clone 2) and original NKL cells (x1000). B, expression patterns of CD2, CD3, CD20, and CD94 were essentially the same among clone 1, clone 2, and original cells.

 
RT-PCR analysis detected transcripts of major lytic genes, BZLF1 and BALF2 in addition to EBER1 and Qp-initiated EBNA1 in both clones (Fig. 8, A and B)Citation . The pattern was the same as that observed at 48 h of EBV exposure. However, Western blot analysis failed to detect ZEBRA and EA-D in these clones (Fig. 9B)Citation . This discordance is probably because of too high sensitivity of nested PCR analysis. In situ hybridization also detected EBERs but not BHLFs in clone 1, which supported the results of Western blot analysis (Fig. 3)Citation . These findings indicated that latent EBV infection but not lytic infection was maintained in these clones.



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  		Fig. 8. Reverse transcription-PCR analysis of EBV-associated genes in EBV-carrying NKL clones. A, EBER1 was expressed in EBV-carrying NKL clones. B, nested PCR analysis detected not only EBNA1 but also BZLF1 and BALF2 in both clones. LMP1 and LMP2A transcripts were positive only in clone 1.

 


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  		Fig. 9. Clone 1 expresses LMP1. A, flow cytometric analysis showed LMP1 expression only in clone 1. Raji and U937 were used as positive and negative controls, respectively. B, clone 1 expresses LMP1 but not EBNA2 in Western blot analysis. Lytic infection markers, ZEBRA and EA-D, were not detected in Raji and the two clones. In EA-D evaluation, nonspecific bands (arrowhead) at upper site of 52 kDa EA-D warrants almost equal amount of protein loading. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-treated Akata was used as a positive control for expression of ZEBRA and EA-D.

 
Although LMP1 and LMP2A transcripts were undetectable during the early phase of EBV infection, we detected them in clone 1 by RT-PCR analysis (Fig. 8B)Citation . Western blotting and flow cytometric analysis confirmed LMP1 expression in clone 1 (Fig. 9, A and B)Citation . Therefore, LMP1 expression should be restricted only after establishment of EBV-carrying NKL clone. LMP1 from clone 1 was apparently <60 kDa on Western blot analysis (Fig. 9B)Citation . The band appeared to be the truncated form, which is usually observed at the early phase of EBV infection. EBNA2 was never detected even by RT-PCR analysis during the entire process of EBV infection. Analysis of various clinical samples including nasal-type NK cell lymphomas has shown the LMP1 expression despite the lack of EBNA2. Here, we demonstrated the same expression pattern of EBV-associated genes in vitro.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Considerable part of NK cell neoplasms possess EBV genome. However, there has been no direct evidence that EBV infects human NK cells. In this study, we used EGFP-EBV to monitor the entry of EBV into human NK cells. Because EGFP signal is generated only after the entry of EGFP-EBV into the target cells, we confirmed EGFP-EBV infection of two NK cell lines and PB-NK cells.

Although it remains unclear whether naive PB-NK cells express the CD21 molecule, our present results showed the lack of CD21 expression in NK-92 and NKL cells. Recent studies have also shown the infection of several human CD21-negative cell lines by EBV (21, 22, 23 , 25) . Janz et al. (24) showed that rEBV lacking gp350/220, a ligand for CD21, infects Raji cells. Although the role of the CD21-mediated pathway during the primary NK cell infection by EBV is controversial, our results support the presence of an unknown virus-cell interaction distinct from the CD21-mediated pathway. HLA class II ß chains are believed to be important for EBV internalization in HLA class II-positive cells (18 , 19 , 39) . Viral glycoproteins, gp42 and gp85, are known to play an important role in EBV internalization into HLA class II-positive cells (21 , 39) . We detected expression of HLA class II on the cell surface of both NK cell lines. Activated PB-NK cells are known to express HLA class II. Therefore, after binding to EBV through a distinct molecule from CD21, both NK cell lines and PB-NK cells may fuse with EBV through the association between the ternary complex and the HLA class II ß chain.

Type I and II latency patterns are recognized in EBV-associated NK cell malignancies (13 , 14) . In the early phase of EBV infection, even RT-PCR analysis detected only EBER1 and Qp-initiated EBNA1 transcripts among the latent genes examined in rEBV-exposed NK-92 and NKL cells. This expression pattern corresponds to type I latency. However, RT-PCR analysis detected LMP1 but not EBNA2 transcript after establishment of NKL-derived clone 1, which corresponds to type II latency. Western blotting and flow cytometric analysis confirmed LMP1 expression in clone 1. Therefore, expression of LMP1 in this clone should be independent of EBNA2. A similar expression pattern of EBV-associated genes was reported in EBV-infected peripheral blood T cells. In the early phase of infection, EBV-infected peripheral blood T cell was reported to lack LMP1 expression (40) . However, established EBV-carrying peripheral blood T-cell clones were shown to express LMP1 (41 , 42) . The temporal profile of latent gene expression pattern during EBV infection in NK cells may be similar to that in peripheral blood T cells.

Although a truncated form of LMP1 is known to appear early after EBV infection or at the lytic phase in B cells (43 , 44) , we detected only the truncated form at the latent phase in clone 1. Detection of shorter sizes of LMP1 was also reported in clinical samples from patients of nasopharyngeal carcinoma (45) . We suppose that some differences at the transcription and translation levels in host cells may lead to the generation of aberrant sizes of LMP1.

In the early phase of infection, we detected BZLF1 and BALF2 transcripts in addition to latent gene expression in rEBV-exposed NK-92 and NKL cells by RT-PCR. Because the results suggested the presence of cells in latent and lytic phases, we additionally evaluated the expression of latent and lytic infection genes by in situ hybridization. In NKL, in situ hybridization showed that lytic and latent phase populations coexisted in EBV-exposed cells after 48 h of exposure. Although RT-PCR analysis detected BZLF1 and BALF2 transcripts even in two established NKL clones, Western blot analysis failed to detect ZEBRA and EA-D proteins in them. In situ hybridization supported this result. Although we cannot fully explain the reason for detection of BZLF1 transcripts, we suppose that expression of BZLF1 may not be so strictly shut-off that nested RT-PCR analysis detected a tiny amount of the transcripts. Therefore, nested RT-PCR analysis alone should not warrant the biologically relevant levels of EBV-associated gene expression. Indeed, nonproducer cell line Raji was reported to express BZLF1 by RT-PCR technique (33) .

We found that some of rEBV-exposed NK cell lines were highly deformed into a bizarre shape. This phenomenon occurred in considerable part of EGFP-positive cells. In situ hybridization showed that NKL cells with abnormal cell shape were usually positive for EBERs’ signal, which suggests that dominant part of EGFP-positive cells showed cell deformation and may enter latent phase of infection. In case of PB-NK cells, cell enlargement in addition to bizarre cell shape was observed after EBV infection. Primary EBV infection of B and T cells was reported to cause Reed-Sternberg cell-like giant cell formation (46) . Furthermore, in situ hybridization demonstrated that giant cells were in latent phase, whereas cells in lytic phase showed a more rounded shape (46) . This study is in good accordance with the present results. EBV-induced giant cells have been believed to be syncytia, which result from the fusion of infected cells (47) . However, Secchiero et al. (48) suggests another possibility of giant cell formation in human herpesvirus-7-infected T cells, i.e., polyploidization with abortive cytokinesis. We suppose that EBV may cause deformation of the infected cell shape through alternation in cytoskeletal components and cell cycle disturbance, including polyploidization. Furthermore, our results showed that most of EGFP-EBV-infected NK-92 and NKL cells enter apoptosis, which explains difficulties in establishing EBV-carrying NK cells in vitro. However, EGFP-EBV enables us to show the EBV infection of human NK cells at an earlier phase than the excursion of apoptosis.

This is the first study clearly showing that EBV infects human NK cells. We observed essentially the same expression pattern of EBV-associated genes in EBV-infected NK-cell lines in vitro as that reported in clinical samples. Although additional studies are required to confirm the critical role of EBV in the development of NK-cell neoplasms, this work may be the first step to elucidate this point.


   ACKNOWLEDGMENTS
 
We thank Dr. Jiang-Hong Gong (University of British Columbia, Vancouver, Canada) and Dr. Michael J. Robertson (Harvard Medical School, Boston, MA) for providing NK-92 and NKL, respectively. We also thank Ayako Okamoto and Keiko Hayashi for technical assistance.


   FOOTNOTES
 
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.

Requests for reprints: Koichi Sugimoto, Division of Hematology, Department of Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.

Received 6/ 6/03. Revised 10/20/03. Accepted 12/31/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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