Biomedical subjects

Kenzo Takada

Publications and source records attributed to Kenzo Takada.

At least 19 recordsLinked to original sources

PubMed2006

Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells.

Epstein-Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C-HT. In the presence of 4HT, LCLs expressed the E3C-HT protein and grew like WT LCLs. When E3C-HT EBV-infected LCLs were transferred to medium without 4HT, E3C-HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C-HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.

B-Lymphocytes↗

PubMed2006

Epstein-Barr virus BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes.

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.

B-Lymphocytes↗

PubMed2006

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.

Animals↗

PubMed2006

EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN.

Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV-infected cells, and are expected to show secondary structures with many short stem-loops. Retinoic acid-inducible gene I (RIG-I) is a cytosolic protein that detects viral double-stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG-I as dsRNA. Transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro-synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. EBERs activated RIG-I's substrates, NF-kappaB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co-precipitated with RIG-I. These results indicate that EBERs are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected cells.

Base Sequence↗

PubMed2006

Involvement of caspase activation and mitochondrial stress in taxol-induced apoptosis of Epstein-Barr virus-infected Akata cells.

Taxol (paclitaxel) is one of the most potent antimicrotubule agents currently used in cancer chemoprevention and treatment. However, the effects of taxol on the induction of apoptosis in Epstein-Barr virus (EBV)-infected cells are unknown. This study investigated the mechanisms of taxol on cell cycle arrest and apoptosis induction using the EBV-infected cell line, Akata. Taxol treatment sensitively and dose-independently induced growth inhibition, cytotoxicity, and apoptosis in the cells, which was demonstrated by the decreased level of tritium incorporation and cell viability, the increased number of positively stained cells in the trypan blue staining and TUNEL assay, the increased population of cells in the sub-G(0)/G(1) phase in flow cytometric analysis, and ladder formation of the genomic DNA. Treatment with z-VAD-fmk almost completely protected the cells from taxol-induced apoptosis indicating that the taxol-induced apoptosis of Akata cells is caspase-dependent. In addition, taxol-induced apoptosis is proposed to be associated with a lower mitochondrial membrane potential and G(2)/M arrest. However, the tubulin expression level doses not appear to be a direct mediator of taxol-induced apoptosis in cells. The presence of EBV in these cells was not related to the sensitivity of the cells to the induction of apoptosis by taxol. Overall, these results demonstrate that taxol induces apoptosis in EBV-infected Akata cells in a dose-independent manner, and that caspase activation and mitochondrial stress are involved in the induction.

Apoptosis↗

PubMed2006

Induction of the early growth response 1 gene by Epstein-Barr virus lytic transactivator Zta.

Early growth response 1 (Egr-1) is a cellular transcription factor involved in diverse biologic functions. Egr-1 has been associated with Epstein-Barr virus (EBV) infection, but it is still unknown whether any EBV protein regulates Egr-1 expression. In this study, we first showed that EBV reactivation is involved in upregulation of Egr-1 and that Egr-1 can be induced by Zta, an EBV lytic transactivator. Zta not only binds to the Egr-1 promoter but also activates the ERK signaling pathway to trigger binding of Elk-1 to the Egr-1 promoter. In addition, knockdown of Egr-1 significantly reduces the spontaneous expression of Zta and Rta in EBV-infected 293 cells, suggesting that a positive-feedback network involving Egr-1 is required for EBV reactivation. This study also implies that Zta has the potential to affect expression of certain genes through Egr-1.

Base Sequence↗

PubMed2006

Epstein-Barr virus (EBV) genome and expression in breast cancer tissue: effect of EBV infection of breast cancer cells on resistance to paclitaxel (Taxol).

The Epstein-Barr virus (EBV) has been detected in subsets of breast cancers. In order to elaborate on these observations, we quantified by real-time PCR (Q-PCR) the EBV genome in biopsy specimens of breast cancer tissue as well as in tumor cells isolated by microdissection. Our findings show that EBV genomes can be detected by Q-PCR in about half of tumor specimens, usually in low copy numbers. However, we also found that the viral load is highly variable from tumor to tumor. Moreover, EBV genomes are heterogeneously distributed in morphologically identical tumor cells, with some clusters of isolated tumor cells containing relatively high genome numbers while other tumor cells isolated from the same specimen may be negative for EBV DNA. Using reverse transcription-PCR, we detected EBV gene transcripts: EBNA-1 in almost all of the EBV-positive tumors and RNA of the EBV oncoprotein LMP-1 in a smaller subset of the tissues analyzed. Moreover, BARF-1 RNA was detected in half of the cases studied. Furthermore, we observed that in vitro EBV infection of breast carcinoma cells confers resistance to paclitaxel (taxol) and provokes overexpression of a multidrug resistance gene (MDR1). Consequently, even if a small number of breast cancer cells are EBV infected, the impact of EBV infection on the efficiency of anticancer treatment might be of importance.

Adenocarcinoma↗

PubMed2006

Epstein-Barr virus infection alters cellular signal cascades in human nasopharyngeal epithelial cells.

Epstein-Barr virus (EBV) latent infection is a critical event in nasopharyngeal carcinoma (NPC) tumorigenesis. EBV-encoded genes have been shown to be involved in immune evasion and in the regulation of various cellular signaling cascades. To elucidate the roles of EBV in NPC development, stable infection of EBV in nasopharyngeal epithelial cell lines was established. Similar to primary tumors of NPC, these infected cells exhibited a type II EBV latency expression pattern. In this study, multiple cellular signaling pathways in EBV-infected cells were investigated. We first demonstrated that in vitro EBV infection resulted in the activation of STAT3 and NFkappaB signal cascades in nasopharyngeal epithelial cells. Increased expression of their downstream targets (c-Myc, Bcl-xL, IL-6, LIF, SOCS-1, SOCS-3, VEGF, and COX-2) was also observed. Moreover, EBV latent infection induced the suppression of p38-MAPK activities, but did not activate PKR cascade. Our findings suggest that EBV latent infection is able to manipulate multiple cellular signal cascades to protect infected cells from immunologic attack and to facilitate cancer development.

Carcinoma↗

PubMed2005

Epstein-Barr virus-encoded small RNA induces insulin-like growth factor 1 and supports growth of nasopharyngeal carcinoma-derived cell lines.

To assess the role of insulin-like growth factor 1 (IGF-1) in the growth of nasopharyngeal carcinoma (NPC), three NPC-derived cell lines, C666-1, CNE1 and HONE1, were examined. C666-1 cells maintained NPC phenotype of Epstein-Barr virus (EBV) expression and were positive for IGF-1 secretion, and their growth was strikingly inhibited by treatment with an anti-IGF-1 antibody under low serum condition. On the other hand, CNE1 and HONE1 cells were EBV-negative and did not secrete IGF-1. Although they could not grow under low serum condition, addition of recombinant IGF-1 made them grow. EBV conversion of CNE1 and HONE1 cells reproduced NPC phenotype of EBV expression and accompanied IGF-1 expression. Although they could grow under low serum condition, their growth was strikingly inhibited by treatment with the anti-IGF-1 antibody. These results suggest that EBV infection induces IGF-1 in NPC cell lines, and that the secreted IGF-1 acts as an autocrine growth factor. These findings seem to be operative in vivo, as NPC biopsies consistently express IGF-1. Further studies demonstrated that increased IGF-1 expression reflected transcriptional activation, and EBV-encoded small RNA (EBER) was responsible for IGF-1 induction. EBER is invariably expressed in EBV-associated malignancies, including NPC. The present findings strongly suggest that EBER directly affects the pathogenesis of NPC.

Cell Line, Tumor↗

PubMed2005

In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2.

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.

Bacterial Outer Membrane Proteins↗

PubMed2005

Epstein-Barr virus (EBV)-encoded BARF1 gene is expressed in nasopharyngeal carcinoma and EBV-associated gastric carcinoma tissues in the absence of lytic gene expression.

The BARF1 gene is located in the BamHI-A fragment of the Epstein-Barr virus (EBV) genome, encodes 221 amino acids, and has activity as an oncogene. Several reports have demonstrated that BARF1 is expressed in the tissues of various EBV-associated epithelioid malignancies. However,BARF1 is thought to be a lytic gene, since its expression is induced upon induction of the lytic cycle in Burkitt's lymphoma cell lines. Therefore, the possibility cannot be excluded that BARF1 expression in EBV-associated epithelioid malignancies reflects spontaneous induction of the lytic cycle in carcinoma cells. The present study aimed to clarify whether BARF1 was expressed as a latent gene or a lytic gene in epithelioid malignancies. Quantitative real-time RT-PCR assay revealed that BARF1 was highly expressed in nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma tissues in the absence of expression of lytic genes. On the other hand, BARF1 protein was detectable only in two of seven NPC tissue samples by immunoblot analysis. Analysis of BARF1-transfected CNE1 cells revealed that BARF1 was quickly secreted into culture medium and was hardly detectable in the cell lysate, which would account for why some NPC tissues were negative for BARF1 protein expression even though they were strongly positive forBARF1 expression at the transcriptional level. The present findings indicate that BARF1 is expressed in NPC and EBV-positive gastric carcinoma tissues as a latent gene and suggest that BARF1 plays a role in the pathogenesis of these malignancies.

Carcinoma↗

PubMed2005

Stable expression of EBERs in immortalized nasopharyngeal epithelial cells confers resistance to apoptotic stress.

Epstein-Barr virus (EBV) infection is closely associated with the development of nasopharyngeal carcinoma (NPC). The EBV-encoded RNAs (EBERs) are the most abundant EBV transcripts (about 10(7) copies per cell) in EBV infected cells. However, the cellular function of EBER expression, particularly in nasopharyngeal epithelial cells, remains poorly understood. EBERs acquire secondary structures analogous to double-stranded RNA (dsRNA) and may bind to the double-stranded RNA-dependent protein kinase (PKR) and interfere with its function. Activation of PKR involves autophosphorylation resulting in protein synthesis inhibition and cellular apoptosis. Induction of cellular apoptosis by activation of PKR may be an antiviral response adopted by virally infected cells. We have examined the functional properties of EBER expression in an immortalized nasopharyngeal epithelial cell line (NP69). Expression of EBERs was achieved by transfecting the NP69 cells with an EBER-expressing plasmid, pESK10. The EBER-expressing NP69 cells attained a higher growth rate compared to cells transfected with control plasmid (pcDNA3). However, the EBER-expressing NP69 cells did not form colonies in soft agar and were non-tumorigenic in nude mice. To investigate if EBERs may protect the nasopharyngeal epithelial cells from apoptotic insults, we treated the EBER-expressing NP69 cells with a dsRNA analogue, poly(I).poly(C) (pIC), to activate PKR in cells and examined for their responses. Lower level of PKR phosphorylation and elevation of Bcl-2 were observed in EBER-expressing NP69 cells. In addition, other apoptotic markers including the cleaved forms of caspase-3 and poly(ADP)ribose polymerase (PARP) were found to be lower in EBER-expressing NP69 cells after treatment with pIC. Lower phosphorylation levels of p38 MAPK (mitogen-activated protein kinase) and c-jun were also observed in EBER-expressing NP cells. Our results suggest that EBER expression may confer an apoptotic-resistant phenotype in immortalized nasopharyngeal epithelial cells.

Animals↗

PubMed2005

Epstein-Barr virus-encoded poly(A)- RNA confers resistance to apoptosis mediated through Fas by blocking the PKR pathway in human epithelial intestine 407 cells.

Our recent findings demonstrated that the Epstein-Barr virus-encoding small nonpolyadenylated RNA (EBER) confers resistance to various apoptotic stimuli and contributes to the maintenance of malignant phenotypes in Burkitt's lymphoma. In this study we investigated the role of EBER in the human epithelial Intestine 407 cell line, which is known to be susceptible to Fas (Apo1/CD95)-mediated apoptosis. Fas, a member of the tumor necrosis factor receptor family, transduces extracellular signals to the apoptotic cellular machinery, leading to cell death. Transfection of the EBER gene into Intestine 407 cells significantly protected the cells from Fas-mediated apoptosis, whereas EBER-negative cell lines underwent apoptosis after Fas treatment. EBER bound double-stranded RNA-dependent protein kinase R (PKR), an interferon-inducible serine/threonine kinase, and abrogated its kinase activity. Moreover, expression of the catalytically inactive dominant-negative PKR provided resistance to Fas-induced apoptosis. Expression of EBER or dominant-negative PKR also inhibited the cleavage of poly(ADP-ribose) polymerase, a mediator of the cellular response to DNA damage, downstream of the Fas-mediated apoptotic pathway. These results in combination indicate that EBER confers resistance to Fas-mediated apoptosis by blocking PKR activity in Intestine 407 cells, consistent with the idea that EBER contributes to the maintenance of epithelioid malignancies.

Apoptosis↗

PubMed2005

Critical role of Epstein-Barr Virus (EBV)-encoded RNA in efficient EBV-induced B-lymphocyte growth transformation.

It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.

B-Lymphocytes↗

PubMed2005

Epstein-Barr virus transforming protein LMP1 plays a critical role in virus production.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.

Cell Nucleus↗

PubMed2004

Epstein-Barr virus-encoded LMP2A regulates viral and cellular gene expression by modulation of the NF-kappaB transcription factor pathway.

Epstein-Barr virus (EBV)-associated malignancies display distinct patterns of virus latent gene expression that reflect the complex interplay between the virus and its host cell. In the EBV-associated epithelial tumor nasopharyngeal carcinoma (NPC), the virus-encoded latent membrane protein LMP2A is consistently expressed whereas the oncogenic LMP1 protein appears to be restricted to only a proportion of tumors. In an attempt to understand the contribution of LMP2A to the pathogenesis of NPC, we established carcinoma cell lines stably infected in vitro with either a wild-type recombinant EBV (rEBV) or a mutant rEBV in which LMP2A is deleted (rEBV-2A). An NPC-like pattern of EBV gene expression including LMP2A but not LMP1 was consistently observed in carcinoma cells infected with rEBV. However, carcinoma cells infected with rEBV-2A expressed high levels of LMP1 from the signal transducer and activator of transcription (STAT)-regulated L1-TR promoter. Consistent with this effect, basal STAT activity was reduced in rEBV-infected carcinoma cells, and this repression was relieved in the absence of LMP2A. This modulation of STAT activity correlated with the ability of LMP2A to inhibit the autocrine secretion of IL-6 from carcinoma cell lines. Exogenous IL-6 was able to induce expression of LMP1 by means of STAT3 activation both in rEBV-infected carcinoma cell lines and in the EBV-positive C666-1 NPC cell line. The LMP2A-mediated suppression of IL-6 was a consequence of NF-kappaB inhibition. These data reveal that LMP2A modulates two key transcription factor pathways in carcinoma cells and suggest that this finding may be important in the pathogenesis of EBV-associated tumors.

Base Sequence↗

PubMed2004

Epstein-Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction.

EBV associates with various T-cell-proliferating diseases such as chronic active EBV infection and nasal lymphoma. In contrast to B cells, which are highly susceptible to EBV infection in vitro, T cells are refractory to EBV infection in vitro, and it has been difficult to examine the effects of EBV infection on T cells. We recently generated EBV recombinants with a selectable marker, which made it possible to select EBV-infected cells even when the efficiency of infection was low. Using the recombinant virus, we found that a human T-cell line, MT-2, was susceptible to EBV infection, and we succeeded in isolating EBV-infected cell clones with type II EBV latency, which was identical with those seen in EBV-infected T cells in vivo. EBV-infected MT-2 cell clones had shorter cell doubling times and higher saturation density than non-EBV-infected counterparts. We found that EBV-positive MT-2 cells expressed higher levels of interleukin (IL)-9 than EBV-negative MT-2 cells at the transcriptional level. It was also demonstrated that EBV-encoded small RNA was responsible for IL-9 expression. Addition of recombinant IL-9 accelerated the growth of MT-2 cells, whereas growth of the EBV-converted MT-2 cells was blocked by treatment with an anti-IL-9 antibody. These results suggest that IL-9 induced by EBV-encoded small RNA acts as an autocrine growth factor for EBV-infected T cells. Analysis of nasal lymphoma biopsies indicated that three of four specimens expressed IL-9. The present findings suggest that EBV directly affects the pathogenesis of EBV-associated T-cell diseases.

Biopsy↗

PubMed2004

Epstein-Barr virus infection of human natural killer cell lines and peripheral blood natural killer cells.

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 approximately 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.

Apoptosis↗