Shinshu Med J, 66⑶：195～204, 2018

Induction of Interleukin-10 in the Stable Transformants of Human
T-Cell Line Expressing Epstein-Barr Virus-Encoded Small RNAs
Junaidi, Ayako Tateishi and Hiroyuki Kanno＊
Department of Pathology, Shinshu University School of Medicine

Background : Epstein-Barr virus (EBV) is a ubiquitous virus in humans and latently infects B cells. In some in­dividuals, however, EBV produces chronic active infection and causes life-threatening complications due
probably to cytokinemia induced by EBV-infected T or natural killer (NK) cells. The role of EBV-encoded small
RNAs (EBERs), which are commonly expressed in EBV-infected T and NK cells, is still unknown.
Methods : The plasmid coding EBERs was introduced into human T-lymphotropic virus-I-negative humanT-cell lines in a site-directed manner, and stable transformants were established. The alteration of cytokine ex­pression in EBERs-expressing transformants and the activation of the downstream signaling cascade fromdsRNA were examined.
Results : Among three mother T-cell lines and their transformants, only the transformants from MOLT-14 cellsγ( δ T cells) expressed EBERs. EBERs-expressing MOLT-14 cells produced a larger amount of interleukin-10
than did the mother cell line. The phosphorylation of dsRNA-dependent protein kinase (PKR) and that of IκBα
which act downstream of PKR, increased in EBERs-expressing clones.
Conclusion : The γδ T cell-specific production of IL-10 through EBERs expression might lead to modification of
the role of γδ T cells, and might play a role in human immune diseases. Shinshu Med J 66 : 195―204, 2018
(Received for publication January 10, 2018 ; accepted in revised form January 22, 2018)
Key words : Epstein-Barr virus (EBV), chronic active EBV infection, EBV-encoded small RNAs,γδ T cell, interleukin-10

Ⅰ Introduction

high viral loads in peripheral blood of the patients1）2）.
CAEBV is frequently associated with the broad

Epstein-Barr virus (EBV) is a ubiquitous virus inspectrum of EBV-associated T/natural killer (NK)-humans. Most individuals are infected with EBV bycell lymphoproliferative disorders including poly­

early adulthood. Primary EBV infection is usually as­

clonal, oligoclonal, and monoclonal proliferation3）,

ymptomatic, but sometimes results in infectious

and is a disease with a high mortality with life-mononucleosis, which is basically self-limited due tothreatening complications, such as virus-associatedthe development of EBV-specific immunity. Howev­

hemophagocytic syndrome, due probably to cyto­er, some individuals in Asia, mostly in Japan, arekinemia induced by EBV-infected lymphoid cells4）,

reported to develop chronic infection with EBV.

EBV-positive apparent leukemia/lymphoma mainly

Chronic active EBV infection (CAEBV) is character­in T- and NK-cell lineage, and cardiovascular dis­ized by chronic recurrent infectious mononucleosis-

eases and arteritis with the infiltration of EBV-

like symptoms over a long period of time and bypositive lymphoid cells1）5-7）.
In CAEBV, the transcripts of EBV genes are de­＊Corresponding author : Hiroyuki Kanno
Department of Pathology, Shinshu University School of
Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621,
Japan
E-mail : hirokan@shinshu-u.ac.jp

No. 3, 2018tected in T or NK cells2）, and the in vitro infection ofhuman T-cell lines with EBV after the enforced ex­pression of EBV receptor in B-lineage cells, CD21,
causes the enhanced production of a macrophage ac­
195

Junaidi・Tateishi・Kanno

tivating cytokine, tumor necrosis factor (TNF)α8）.

Center, Hayashibara Biochemical Labs., Inc., Okaya­

However, the EBV gene responsible for the upregu­

ma, Japan), and an EBV-positive Burkitt’s lymphoma

lation of TNFα expression in T-lineage cells remains

cell line, Akata (a gift from Prof. Kenzo Takada ;unclear. Profiles of EBV latent gene expression

Hokkaido University, Sapporo, Japan), were grownin EBV-infected T and NK cells show variations,in RPMI 1640 medium supplemented with 10 %and only EBV nuclear antigen-1 (EBNA1) andheat-inactivated fetal bovine serum (Cambrex Bio

EBV-encoded small RNAs (EBERs) are commonly

Science, Walkersville, MD). An EBV-positive humanexpressed in non-neoplastic infected T and NK cells

NK cell line, SNK6 (a gift from Dr. Norio Shimizu ;9）in CAEBV . EBNA1 enables efficient EBV episome

Tokyo Medical and Dental University, Tokyo, Japan),

replication, transcription, and maintenance in la­

was grown in Artemis-2 medium (Nihon Techno

10）

tently infected dividing cells . Recently, EBNA1 wasService, Ushiku, Japan). ZeocinTM and hygromycin B

shown to stably interact with a ubiquitin-specific prote­

used for the selection of drug-resistant clones werease called USP7 and to protect cells from apoptotic

purchased from Invitrogen (Carlsbad, CA). A humanchallenge by lowering the p53 level11）. However,cervical carcinoma cell line, Hela, a human mammary

EBNA1 has been shown to activate transcrip­t ion

carcinoma cell line, MCF7, and NIH3T3 were ob­from episomal but not integrated DNA, and would

tained from the Health Science Research Resources12）

not affect the cellular gene expression . Therefore,

Bank (Sennan, Japan), and cultured in Eagle’s mini­

among EBV latent genes expressed in T and NK

mal essential medium with 10 % heat-inactivatedcells in CAEBV and EBV-associated hemophagocyt­

fetal bovine serum.

ic syndrome patients, EBERs may cause the activa­

B

Plasmids

tion of T and NK cells including the induction of

Flp recombinase-mediated integration kit, Flp-InTM

cytokines, which would be responsible for the devel­

System (including vectors, pFRT/lacZeo, pOG44, andopment of the diseases. EBERs cause interleukin

pcDNA5/FRT), was purchased from Invitrogen. The(IL)-10 induction in human B-lineage Burkitt’s lym­

1.0 kb SacI-EcoRI subfragment from the EcoRI K

13）phoma cell lines . Furthermore, EBV infects a hu­

fragment (EKS) of Akata EBV DNA, which corre­

man T-lymphotropic virus-I (HTLV-I)-positive hu­

sponds to the EcoRI J fragment of B95-8 EBV DNA

14）

man T-cell line, MT-2 , and EBERs induce IL-9 in
15）

and contains EBER1 and EBER2 reading frames,

EBV-infected MT-2 cells . However, the HTLV-

was amplified, and BglII and KpnI sites were added

I infection activates human T cells and induces vari­at each end of the fragment with polymerase chain

16）

ous cytokines , thus the alteration of cytokine ex­reaction (PCR)7）. The EKS fragment was subcloned

pression in HTLV-I-positive T-cell lines with theinto the BglII and KpnI sites, between those cyto­

superinfection of EBV might not reflect the role ofmegalovirus promoter sequence locates, in pDNA5/

EBV in vivo. In this context, the role of EBERs in

FRT vector (pDNA5/FRT-EKS). The sequence of

HTLV-I-free human T-cell lines is still unknown.

the insert was confirmed to be the same as the re­In the current study, we introduced the plasmid
coding sequences of EBERs into HTLV-I-negative

ported EBV sequence.
C

Transfection and selection of transformants

human T-cell lines in a site-directed manner, estab­

Transfection-grade plasmid DNA was purified

lished stable transformants, and examined the rolewith a FlexiPrep kit (GE Healthcare, Uppsala, Swe­of EBERs in human T cells.

den). Plasmids were introduced to T-cell lines by

ⅡA

Materials and Methods

Cell lines and culture

the electroporation method. 5×106 cells were sus­pended in serum-free RPMI 1640, washed twice, andresuspended in 600 μl of ice-cold serum-free RPMI

HTLV-I-negative human T-cell lines, Jurkat,

1640 containing 20 to 100 μg of plasmid DNA in a 4-

MOLT-4, and MOLT-14 (a gift from Fujisaki Cell

mm-gap electroporation cuvette. The electroporation

196

Shinshu Med J

Vol. 66

IL-10 Induction by EBERs in T-Cell Linewas performed with the Gene Pulser II (Bio-Rad,ly described7）13）. The 315 bp-fragment of human actin

Hercules, CA) at 1 mF, and the optimal voltage formRNA was amplified with reverse transcriptase

each cell line. The voltage at electroporation was(RT)-PCR as previously described7）18）. After PCR

optimized for each cell line by using pEGFP-C1 andamplification, the amplified segments were electro­

percent fluorescence-positive cells as an indicator.

phoresed in agarose gels, cut out, purified with theAfter electroporation, cells were cultured for 2 days

JETSORB kit (GENOMED, Bad Oeynhausen, Ger­

4

without selection reagents, and subsequently 2×10many) and used as templates for probe labeling reac­

cells were seeded in a well of flat-bottomed 96-welltion.

culture plates with 200 μl of selection medium. Half

G

Real time RT-PCR analysesof the medium was changed every 5 days until colo­

Real time RT-PCR analyses were performed us­

nies emerged. Clones were expanded and maintained

ing the TaqMan assay system (Applied Biosystems,

in selection medium.

Foster City, CA) as previously described19）. Pre-D

Recombinant cytokines and other reagentsdeveloped TaqMan assay reagents used in this study

Recombinant human interferon (IFN)α (rhIFNα ;were #NM 000575 for human IL-1α, #NM 000576 for8

1.8×10 U/mg protein), recombinant human IL-1α9(rhIL-1α; 1×10 U/mg protein) and recombinant
7

human IL-1β, #NM 000589 for human IL-4, #NM
000600 for human IL-6, #NM 000572 for human IL-

human TNFα (rhTNFα ; 2×10 U/mg protein) were10, #NM 000594 for human TNFα, #NM 000619 forpurchased from PeproTechEC (London, UK). Calyculin

human IFNγ, and #NM 002046 for human glyceral­

A was purchased from Sigma-Aldrich (St. Louis,

dehyde 3-phosphate dehydrogenase (GAPDH ; all fromMO).

Applied Biosystems). Serial ten-fold dilutions of theEsample with the lowest threshold cycle were used toSouthern blot anaysis
DNAs were extracted from cell lines, quantified

obtain the standard curve of relative amounts ofby measuring optical density (OD)260 and stored ateach mRNA. For the real time RT-PCR analyses of－20 ℃ until use. Aliquots of 10 μg of DNA were di­

EBER1 and EBER2, total RNA was treated withgested with HindIII, electrophoresed in 0.6% agarose

DNase using TURBO DNA-freeTM kit (Invitrogen),gels and Southern blotted as previously described17）.

and converted to cDNA with reverse-transcription.

The 230 bp PvuII/NcoI fragment of pFRT/lacZeo

Primers and TaqMan probes were designed with

was used as a template. The nonradioactive probe

Primer Express (v.1.5 ; Applied Biosystems). Primer

was labeled by random prime labeling, Southern-sequences were 5’-GTGAGGACGGTGTCTGTGGTT-3’

blotted filters were hybridized with the labeled

and 5’-TTGACCGAAGACGGCAGAA-3’ for EBER1,

probe, and the signals were generated by chemilu­

and 5’-GCTACCGACCCGAGGTCAA-3’ and7）

minescence as described previously .

5’-GAGAATCCTGACTTGCAAATGCT-3’ forF

RNA extraction and Northern blot analysis

EBER2, amplifying the 58 bp and 77 bp segment of

Total RNA was extracted from cell lines using

RNA, respectively. TaqMan probe sequences were

Trizol reagent (Invitrogen), quantified by measuring

5’-TCTTCCCAGACTCTGC-3’ for EBER1 andOD260, and ethanol-precipitated at －80 ℃ until use.

5’-AAGAGAGGCTTCCCGCC-3’ for EBER2. Plasmid

Twenty μg of total RNA was electrophoresed incontaining the EKS fragment of EBV DNA, which

1.5 % formaldehyde denaturing agarose gels, andcontains EBER1 and EBER2 reading frames, was

17）

Northern blotted as previously described . Hybrid­

used as standard DNA samples for absolute quantifi­

ization with nonradioactive DNA probes and signal

cation of copy numbers of EBERs.

generation were performed in the same manner as

H

Measurement of IL-10

described in the previous section. The full sequence

The amounts of human IL-10 in culture super­of EBER1 (167 bp) was amplified with PCR using thenatants of cell lines were quantitated by enzyme-

DNA sample extracted from Akata cells as previous­

linked immunosorbent assay (ELISA) using humanNo. 3, 2018197

Junaidi・Tateishi・Kanno

IL-10 ELISA kit (R&D systems Inc., Minneapolis,

and the plasmid-integrated clones were selected inMN).

the culture containing Zeocin at a concentration ofI

100 μg/ml. After the establishment of several clones

Immunoblot analyses
Cells were lysed and cell lysates were separated

from each mother cell line, clones with a single FRTwith sodium dodecyl sulfate-polyacrylamide gelsite were selected with Southern blot analyses, andelectrophoresis and transferred to membranes, as

we obtained two clones with a single FRT site in20）

described previously , with the exception of adding

each mother T-cell line. We next transfected 10 μg

phosphatase inhibitors (1 mM sodium orthovanadate,of pcDNA5/FRT-EKS containing EBERs sequence

30 mM tetrasodium pyrophosphate and 50 mM sodi­

and 90 μg of pOG44 plasmids into T-cell clones withum fluoride) to the buffer for cell lysis. Membranes

a single FRT site by electroporation, and the cells

were blocked and incubated with primary antibody

with the recombination at the FRT site integrating

solutions, and signals were generated by chemilumi­

pcDNA5/FRT-EKS plasmid were selected in the20）

nescence, as described previously , and stored asculture containing hygromycin at a concentration ofdigital images using an image analyzer (LAS-4000

200 μg/ml. After the establishment of hygromycin-mini ; GE Healthcare). Antibody solutions used in thisresistent cultures, the integration of the plasmid instudy were mouse monoclonal antibodies to β-actin

the manner of site-directed recombination was con­(Sigma-Aldrich), IκBα and phospho-IκBα (Ser32/36),

firmed by Southern blot analyses (Fig. 1). Then, weand rabbit polyclonal antibodies to dsRNA-depend­

examined the expression of EBER1 in the estab­ent protein kinase (PKR), phospho-PKR (Thr451),

lished transformants by Northern blot analyses.

and IL-1 receptor-associated kinase-1 (IRAK-1)

Among three mother T-cell lines and their transfor­(all from Cell Signaling Technology Inc., Beverly,mants, only the transformants from MOLT-14 cellsMA) diluted with 5 % bovine serum albumin in Tris-

expressed EBER1 (Fig. 2). The amount of EBER1

buffered saline containing 0.1 % Tween 20, andexpressed in MOLT-14 clones was, however, far lessrabbit polyclonal antibody to phospho-IRAK-1 (Ser376)

than that in an EBV-positive Burkitt’s lymphoma(Abgent, San Diego, CA) diluted with Can Get Signal

cell line, Akata. Furthermore, we quantitated the

Solution-1 (Toyobo, Osaka, Japan).

copy number of EBERs transcripts by real time RT-J

PCR analyses. In MOLT-14 clones, the amount of

Statistical analysis
Statistical significance was determined by the two-

total EBERs transcripts was approximately 2 % oftailed Student’s t-test. p＜0.01 was considered sta­

those of an EBV-positive NK cell line, SNK6 (Table

tistically significant.

1). EBER1 transcripts in MOLT-14 clones wereⅢ

A

Results

Establishment of stable EBERs-transformantsof T-cell lines
To access the exact effects of EBERs in T-cell lines,

much lower than those in SNK6, although EBER2transcripts in MOLT-14 clones were higher thanthose in SNK6 (Table 1).
B

Alteration of cytokine expression in MOLT-14
cloneswe employed the site-directed recombination sys­

The expression of cytokines from EBERs-expressing

tem. By comparing the expression of cytokines

MOLT-14 cells, which might activate macrophage-

between T-cell clones with the FRT site alone andlineage cells and affect immune conditions, wasthose with EBERs sequence integrated in the same

examined at the mRNA level, at first.

FRT site, we could rule out the effects of the inte­
gration site in the genome.

By real time RT-PCR analyses, mRNA expressionof IL-10 in EBER-expressing MOLT-14 cells was

At first, we transfected 20 μg of ScaI-cut pFRT/

much higher than that in mother cell lines with alacZeo plasmids into the three human T-cell lines,

FRT site (Table 2). The expression of IL-4 and TNF

Jurkat, MOLT-4, and MOLT-14, by electroporation,

α mRNA was not apparently affected by the expres­198

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IL-10 Induction by EBERs in T-Cell Line

Fig. 1 Southern blot analyses for the integration of plasmids. Ten micrograms of DNA was digested
with HindIII, electrophoresed in 0.6 % agarose gels, Southern blotted, and hybridized with DNA
probe containing SV40 promoter sequence derived from integrated pFRT/lacZeo plasmid, andsignals were visualized as described in “Materials and Methods”. DNA size markers are indicatedon the left. DNA samples from each FRT clone contain a single band, and those from FRT
recombinant clones with pDNA5/FRT-EKS (EBER clones) exhibit the shift of a band to a larger
size, compatible with the size of the plasmid (approximately 5 kb larger). (－), mother cell line ; FN,
clones containing a FRT site ; FNE, clones containing EBERs sequences with FRT recombination.

Fig. 2 Northern blot analyses of EBER1 expression in T-cell clones. Human T-cell clones were
cultured for at least 7 days in the absence of selection antibiotics. Twenty micrograms of total
RNA samples were applied to each well, Northern blotted, and hybridized with nonradioactive
EBER1 and actin DNA probes, and signals were visualized as described in “Materials andmethods”. MOLT-14 clones express EBER1 at the level detectable with Northern blot, but Jurkat
and MOLT-4 clones do not. RNA samples from Akata cells (Ak) served as positive control
samples. J, Jurkat ; (－), mother cell line ; FN, clones containing a FRT site ; FNE, clones containing
EBER sequences with FRT recombination.

sion of EBERs (Table 2). The expression of IL-1α,
IL-1β, IL-6 and IFNγ mRNA in these cells was ex­

Table 1 Expression of EBER1 and EBER2 in cell lines
analyzed by real time RT-PCR

tremely low, that is to say, the threshold cycles ofthese mRNAs were over 33 cycles with much devia­tion, and an exact comparison of expression level
was not possible (results not shown). The change of
IL-10 mRNA expression in Jurkat and MOLT-4 celllines was not obvious after the integration of EBERsNo. 3, 2018

Copy number (copy/ng total RNA)
Cells

EBER1

EBER2

Total

F7E

3

1.34 × 10

3.64 × 10

21.70 × 103

F8E

1.06 × 103

3.85 × 102

1.45 × 103

SNK67.26 × 104

1.14 × 102

7.27 × 104
199

Junaidi・Tateishi・Kanno

containing plasmid without EBERs expression (re­Table 2 Real time RT-PCR analyses of the expressionof cytokines in EBERs-expressing MOLT-14 clones

sults not shown). The increased IL-10 protein in 3-day
culture supernatants of EBERs-expressing MOLT-14
cells, compared with that of mother cell lines, wasconfirmed by ELISA (Fig. 3).
C

Increased phosphorylation of PKR and IκBα in
EBER-expressing MOLT-14 clones
To demonstrate that the EBERs expressing in

MOLT-14 cells were actually functioning, we exam­
ined whether the downstream signaling cascade fromdsRNA was activated by immunoblot analyses for the

Cells

IL-4
＊

F7E

0.43 ＋/－ 0.06

F8E

0.62 ＋/－ 0.02

IL-10

TNFα

28.2 ＋/－ 13.0

1.45 ＋/－ 0.46

6.64 ＋/－ 1.01

1.83 ＋/－ 0.17＊

Relative expression level of mRNA compared to thatin the mother line containing the FRT site alone, which
are standardalized with the expression of mRNA of
GAPDH. The data are presented as the mean value of
three independent experiments plus/minus standard
deviation.

phosphorylated signaling proteins involved in the cas­cade.
PKR is the first-described dsRNA sensor mole­cule, and is phosphorylated and activated with theviral dsRNA in infected cells21）. We thus examinedat first the alteration of phosphorylation of PKR with
EBERs expression. The phosphorylation of PKR in­
creased along with the expression of EBERs in ourT-cell system (Fig. 4A), although the protein ex­pression of PKR was not clearly changed (Fig. 4A).
To confirm the increased activation of PKR in EBERsexpressing T-cell clones, we next examined the phos­
phorylation of IκBα, which acts downstream of PKR
in the signaling cascade22）, and revealed a slightly in­creased phosphorylation of IκBα in EBERs-expressing
clones without alteration of protein expression(Fig. 4B).
Furthermore, to evaluate the involvement of in­nate immune system in the EBERs-induced IL-10
expression, we next examined the phosphorylationof IRAK-1, which is a critical signaling mediator of

Fig. 3 Concentration of IL-10 protein in the culture of
MOLT-14 clones. After the 3-day culture of celllines, the supernatants were harvested by centrifu­
gation and stored at －80 ℃. The concentration of
IL-10 was measured by ELISA. The bars represent
the mean values of triplicate from two independent
supernatant preparations plus/minus standard devi­
ation.
＊

P＜0.01. Mo., mother cell line ; FN, clones containinga FRT site ; FNE, clones containing EBERs sequenceswith FRT recombination.

innate immunity23）and is involved in the LPS-induced

formants of MOLT-14 cells expressing EBERs andIL-10 expression24）, by immunoblot analyses. MOLT-

revealed the increased production of IL-10 from these

14 clones containing a FRT site varied in protein

EBERs-expressing MOLT-14 cells.

expression and phosphorylation of IRAK-1 and the

The expression of EBERs in MOLT-14 clones waschange induced by the expression of EBERs was notmuch lower than that of an EBV-positive NK cell

uniform in these two clones (Fig. 4C). One clone (F7)line, SNK6, and the ratio of EBER1 to EBER2 in MOLT-

showed up-regulated expression of IRAK-1 protein

14 clones was greatly different from that in SNK6.

following EBERs expression and the other (F8) showedThis is not surprising because these variations in

down-regulated expression (Fig. 4C).

EBERs expression were reported in EBV-positive

Ⅳ Discussion
In the current study, we established stable trans­
200

B-lineage cell lines25）. Furthermore, EBER2 induces
IL-6 and plays a role in EBV-induced B-cell growthtransformation dominantly over EBER126）. Therefore,
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IL-10 Induction by EBERs in T-Cell Line

A

B

C

Fig. 4 Expression and phosphorylation of signaling molecules, such as PKR (A), IκBα (B), and IRAK-1 (C), in
MOLT-14 clones. MOLT-14 clones were cultured for at least 7 days in the absence of selection antibiotics.
Subconfluent cultures of Hela, NIH/3T3, and MCF7 cells were incubated with rhIFNα (1,000 U/ml for 18hrs) and subsequently with calyculin A (100 nM for 15 min), with rhTNFα (1,000 U/ml for 10 min), andwith rhIL-1α (20 ng/ml) and calyculin A (100 nM) for 5 min, respectively. Cells were harvested, 500 μl ofcell lysates were prepared from 5×106 cells, and a 10-μl (for β-actin) or 30-μl (for signaling molecules) of
aliquot of each sample was applied to each well. After electrophoresis in 12 % (β-actin and IκBα) or 10 %(PKR and IRAK-1) polyacrylamide gels, proteins were transferred to a polyvinylidene difluoride mem­brane, incubated with the antibodies, and visualized as described in “Materials and methods”. (－), mothercell line ; FN, clones containing a FRT site ; FNE, clones containing EBERs sequences with FRT recombina­
tion ; H, Hela ; HI, Hela treated with IFNα and calyculin A ; T, NIH3T3 ; TT, NIH3T3 treated with TNF
α; M, MCF7 ; MIL, MCF7 treated with IL-1α and calyculin A.

even the lower expression of total EBERs with con­

The induction of IL-10 in T-lineage cells by EBERs

siderable expression of EBER2 may play a role inis compatible with the increased IL-10 in the sera of

the induction of an immune-modulating cytokine,

patients with T-cell type CAEBV2）, although EBERs

IL-10.

did not induce a cytokine with macrophage-activat­

No. 3, 2018201

Junaidi・Tateishi・Kanno

ing activities, such as TNFα, IFNγ, and IL-1β. MOLT-

Increased phosphorylation of IκBα (Fig. 4B) indi­

14 is a T-cell receptor γ/δ-bearing human T-cellcates increased transcription of the genes controlled

line and exhibits NK-like cytotoxic activity to K562

by nuclear factor κB, which includes a variety ofcells after treatment with rIL-2 or phorbol myristatecytokines, but does not explain the increased cytokine

acetate27）, and thus, shares the characteristic features

expression specific for IL-10. As a candidate for2）of EBV-infected cells in CAEBV patients . Therefore,

the IL-10-specific regulatory molecule, we evaluated

MOLT-14 cells would be suitable for the model cells

the possible involvement of IRAK-123）24）. However, theof functional analyses of EBV genes. On the otherprotein expression and phosphorylation of IRAK-1 werehand, Jurkat and MOLT-4 cell lines are basicallynot associated with EBERs expression and subsequent28）

T-cell receptor α/β-bearing human T-cell lines ,IL-10 induction (Fig. 4C). Thus, the involvement29）

although MOLT-4 lacks the T-cell receptor α gene .

of the innate immune system in this process23） is

Therefore, the cell line-dependent expression of EBERs

uncertain and the mechanism for the specific up-

in T-lineage cell lines (Fig. 2) might be useful for

regulation of IL-10 is still unclear.

the analysis of transcription mechanisms of EBERs.

The induction of IL-10 by EBERs is common to both

PKR is phosphorylated and activated with theB-13） and T-lineage cells. IL-10 has two functions inviral dsRNA in infected cells21）. As for the interaction

the immune system, inhibitory effects on cellularwith EBERs, however, the activation of PKR is in­immunity, such as the inhibition of cytotoxic T lym­

30）

hibited by EBERs , and in Burkitt’s lymphoma cell

phocyte induction, and accelerating effects on humoral

lines, EBERs induce the inactivation of PKR and

immunity, such as the promotion of B cell growth31）

cause resistance to IFN-induced apoptosis . Thus,and differentiation. In the latter aspect, IL-10 seems

the increased phosphorylation of PKR along with theto be associated with human autoimmune diseases,

expression of EBERs in our T-cell system (Fig. 4A)

such as systemic lupus erythematosus34）and auto­

is not consistent with these previous reports. On the

immune hemolytic anemia35）. Since EBERs are non-other hand, Ruf et al reported that EBERs do notpolyadenylated, untranslated RNAs and subsequently

inhibit PRK during the protection from IFN-induced

escape from host immune responses, continuous

32）

apoptosis . Furthermore, the low-level expression

stimulation of B-cell growth would be permittedof HIV-I TAR RNA causes the phosphorylation andthrough the development of diseases. Therefore,

activation of PKR, although the higher expression of

although EBERs in T-lineage cells did not seem to

33）

the same RNA inactivates PKR . Thus, the low-play a role in the development of hemophagocytic

level expression of EBERs observed in the current

syndrome, which is a major complication of CAEBV,

T-cell system would cause the phosphorylation of

the induction of IL-10 by viral small RNAs, such as

PKR, like HIV-I TAR RNA, but anyway, the dose-

EBERs, might play a role in the development ofdependent effects of EBERs on the activation of PKR

human immune diseases with B cell hyper-reactivity.

must be clarified.
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