Journal of General Virology (1996), 77, 1163-1168. Printed in Great Brita#z 1163

Short communication

Transcriptional analysis of the Epstein-Barr virus interleukin-10 homologue during the lytic cycle

Robert Touitou, Chantal Cochet and Irene Joab*

C N R S U R A 1301, Inst i tut Gustave Roussy , 39 rue Camil le Desmoulins , 94800 Villejuif, France

The Epstein-Barr virus (EBV) open reading frame (ORF) BCRF1, expressed in the late phase of the viral cycle, encodes a homologue of human intedeukin-10 (hlL-10). Unspliced, 3' co-terminal transcripts of 0-8 and 1.6kb from O-tetradecanoylphorbol 13-acetate (TPA)-treated B95-8 cells have been described but other results indicated the existence ofuncharacterized trans-

cript(s) initiated upstream of the 1.6 kb BCRF1 mRNA. Here we describe two additional large transcripts of the B C R F 1 0 R F , a possibly spliced product of 3-5 kb and an unspliced product of 4.5 kb. The time course of the expression of BCRF1 transcripts and of the secreted protein from Akata cells were also determined.

The product of Epstein-Barr virus (EBV) open reading frame (ORF) BCRF1 which is expressed in the late phase of the viral life cycle, is 84 % homologous at the amino acid level with mature human interleukin-10 (hIL-10) (Vieira et al., 1991). This cytokine has been described as an immunoregulatory protein which could play a major role as a suppressor of immune and inflammatory responses (for a review see Moore et al., 1993). BCRF1 protein, also termed viral IL-10 (vIL-10), inhibits the synthesis of cytokines [interferon (IFN)-? and IL-2] by activated T helper 1 cell clones (TH1) and induces both the ctonal expansion of activated human B cells and their maturation into Ig-secreting cells (Rousset et al., 1992). All these properties might enhance viral survival in the host, since EBV infects B cells and IFN-7 is a major host defence against EBV transformation of B cells (Lotz et al., 1985).

Unspliced transcripts of the B C R F 1 0 R F from O- tetradecanoylphorbol 13-acetate (TPA)-treated B95-8 cells, have been described (Hudson et al., 1985; Lau et al., 1993): a 0"8 kb mRNA (which could be initiated from the BCR1 promoter) and a 1.6 kb transcript. Both mRNAs have a common 3' end. Results reported by Lau et al. (1993) indicated the existence of uncharacterized transcript(s) initiated upstream of the 1"6 kb BCRF1 transcript. Besides, a report from Stewart et al. (1994) described the expression of an additional 1 kb BCRF1 transcript from induced B95-8 and Akata cells.

A 2-ZAP cDNA library (Stratagene) was prepared using poly(A) + RNA from TPA-treated B95-8 cells. This

* Author for correspondence. Fax + 33 1 45 59 70 09. e-mail ijoab@igr.fr

library was screened with a probe covering the whole BCRF1 sequence. Four polyadenylated cDNAs (F1, F2, F3 and F4) were isolated. Sequencing of approximately 100 bp of both 5' and 3' ends and restriction analysis of the four cDNAs were performed; they differed only in the extent of their 5' region. The cDNAs ranged respectively from 9247, 8843, 8770 and 8561 to 10280 and were collinear with the EBV genome (Fig. 1 a). All the cDNAs contained poly(A) tracts at the Y end, confirming that they were derived from mRNAs; they may represent partial copies of BCRF1 transcripts. Polyadenylation occurs 18 bases after the consensus site AATAAA confirming the observation of Hudson et al. (1985). All these cDNAs were unspliced and presented the same 3' end which has already been determined for the 0'8 and 1.6 kb mRNAs. The F4 cDNA (1719 bp) is longer than the other BCRF1 mRNAs previously described (Hudson et al., 1985; Lau et al., 1993). The 5' end (8561) of this cDNA was located upstream of the initiation point (8708) of the 1.6 kb mRNA described by Lau et al. (1993).

Transcription of BCRF1 gene was investigated by Northern blotting. Northern blotting was performed with total RNA from Akata cells (RNAzol B method; Biotecx Laboratories) treated with rabbit anti-human IgG antibodies (1% v/v; Dakopatts A424). Akata cells were chosen because the disruption of latency by anti- human IgG antibodies treatment is followed by a dramatic and synchronous increase in cells positive for lytic antigens (Takada & Ono, 1989). The hybridization with a complementary single-strand uniformly labelled [~-32P]dCTP DNA probe covering the B C R F 1 0 R F (1AS, 10229 to 9656 on the B95-8 EBV map) revealed

0001-3729 © 1996 SGM

1164 R. Touitou, C. Cochet and I. Joab

(a)

21 x30bp repeats

I 7420 oriP

8561

(

8708

I

8770

(

65 bp palindrome

I 9082

8843

(

BCR1 9631 IIII

9662

I

9247

L_

ATG 9675

r

1.6 kb mRNA

BCRF1

0-8 kb mRNA

F4

F1

F2

F3

10184

[ (Hudson et al., 1985)

IP I (Lau et al., 1993)

(A)18

(A)18

(A)>100

(A)2o 1719 bp

10280

(b)

q

4AS

oriP

(c)

('iv)

2 4 1 5 6 0

N

I I 7420 8042 9082

FR DS

~ g g ~ g

3AS 2AS

(iii)

2415 6 0 . . . . .

( i i )

2 4 1 5 6 0

AATAAA BCR1

== r BCRF1 m l k ,

it

1AS

(i) 2 4 1 5 6 0

kb 4"5 kb---i I i~'e~°':ii : - 4.4 3.5 kb-,--{ ~g:

{ ~ - 2.37 1-6 kb " - i

- 1.37

0.8 kb -,'--i

Fig. 1. (a) Isolation of BCRF1 polyadenylated cDNA from a library constructed from TPA- treated B95-8 cells. The structures of the four BCRF1 cDNA (F1, F2, F3 and F4) are shown. All co-ordinates are given relative to the B95-8 EBV map (Baer et al., 1984). Full boxes indicate both 5' and 3' ends which were sequenced. The 21 x 30 bp repeat, the 65 bp palindrome contained in the orz~ region, the origin for plasmid rep- lication (Yates et aL, 1984) are shown. The BCRF1 reading frame (9675 to 10184), preceded by a potential BCR1 promoter (9631) and followed by a potential polyadenylation signal (10257) are represented. The BCRFI transcripts (0"8 kb and 1.6 kb) described respectively by Hudson et al. (1985) and Lau et aL (1993) are also shown. (b) The structure of the 1AS, 2AS, 3AS and 4AS single-strand DNA probes are repre- sented on the EBV genome with their B95-8 co- ordinates. (c) Northern blotting experiments. The probes used were (i) 1AS, (ii) 2AS, (iii) 3AS and (iv) 4AS ssDNA. The lanes are for total RNA (30 gg/track) from Akata cells treated with rabbit anti-human IgG. The times after induction (h) are indicated above each lane. The sizes of the BCRF1 mRNAs in kb are indicated at the left. The numbers indicated at the right refer to the sizes of the RNA markers.

Expression of viral IL-IO in EBV-infected cells 1165

(a)

bp

600

500

400

t q)

Akata

0 9 15

Fig. 2. (a) S1 mapping 5' end of the 4.5 kb BCRF1 transcript. The ssDNA probe used was 5' 3Zp-labelled AS (6141-5752). The lanes are for undigested probe (P), yeast tRNA control (t), ssDNA phagemid control containing EBV sequence (5174-7314) (q~) and total RNA (30 lag/track) from Akata cells treated with rabbit anti-human IgG for 0, 9 and 15 h. (b) S 1 mapping results are summarized. The large late transcript encompassing BNRFI ORF responsible for the EBV full- length protected fragment of 390 bases and the 5" end of the 4-5 kb late BCRF1 transcript responsible for the protected fragment of 280 bases are shown. The sequence located upstream of the 4.5 kb late BCRF1 transcript start point is represented. The putative TATA box sequence is underlined.

300

280 bp

200

(b) TGA AATAAA 5690 j 5 8 4 1

BNRF1

7420 8042

m ~

CCTCCC C CC C fig FIC CTGTGTGCTGIATTTFIC AflFIC A CTAC AATAA flCC CAflTGTGC AFIATGIGGI-I-[GTfl

5800 5860

Large late transcript encompassing BNRF10RF

,Ip,- ? (Hudson et al., 1985)

5860

( 4.5 kb late BCRF1 transcript w

/ 5752

6141 3 2 * (5' P-labelledAS Probe) 450bases

• (EBV full-length protection) 390 bases

5860 (5' end protection) 280 bases

1166 R. Touitou, C. Cochet and L Joab

(a)

kb 0 3 6 9 12 15 18 21 24 kb

9-49 7"46

4.4 4.5 3"5

2"37 1"6

1-37

0"8

(b)

kDa . m , ~ N ~

14 --;: { vlL-10

0-24

0 3 6 9 12 15 18 21 24

~ } ~ e "~ ~ i ~ i ~ } ~ ~::~li~i: ~ HGPRT

Fig. 3. (a) Northern blotting experiment. The probe used was 1AS (10229 9656) (see Fig. 1 b). The lanes are for total R N A (30 gg/track) from Akata cells treated with rabbit an t i -human IgG. The times after induction (h) are indicated. The sizes of the BCRF1 m R N A s in kb are indicated at the right. The transcripts of the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) housekeeping gene are shown below. (b) Western blotting experiment. Samples corresponding to 2 x 106 cells were removed at the indicated times after induction (h) and supernatants were immunoprecipitated and Western blotted with a rabbit anti-BCRF1 polyclonal serum (499). The numbers indicated at the left refer to the sizes of the molecular mass markers.

four major bands migrating with 0.8, 1.6, 3-5 and 4.5 kb RNAs [Fig. 1 c, panel (i)]. Treatment by phosphonoacetic acid (PAA) (200 gg/ml) considerably diminishes the level of expression of these transcripts, showing that they correspond to copies of late genes (data not shown). Northern blotting performed with poly(A) + RNA ob- tained from TPA-treated B95-8 cells exhibit the same pattern. Nevertheless, the bands were less visible (data not shown).

To characterize the 5' regions of the 3-5 and 4.5 kb BCRF1 transcripts, we performed Northern blotting using probes located upstream of the 1.6 kb BCRF1 transcript. The 2AS probe (8468 to 8112), located just downstream of the Family Repeat (FR) sequence (represented in Fig. 1 b), hybridized with the larger (3.5 and 4.5 kb) BCRF1 transcripts [Fig. 1 c, panel (ii)]. On the contrary, the 3AS (8029 to 7447) and 4AS (7418 to 7032) probes, located respectively within and just upstream of the FR, failed to hybridize with the 3"5 kb transcript [Fig. 1 c, panels (iii) and (iv) respectively]. Hybridization with the 3AS probe, lying within the FR region [Fig. l c, panel (iii)] exhibited a weak signal probably due to the structure of this sequence. The lack of detection of the 3-5 kb transcript with the 3AS probe is unlikely to be due to difficulties of hybridization with this probe, since the 4.5 kb transcript which was less abundant than the 3.5 kb one [Fig. 1 c, panels (i) and (ii)], was clearly detected [Fig. 1 c, panel (iii)]. This might indicate that the 3.5 kb transcript undergoes at least one splicing event.

The 4-5 kb transcript remained visible not only with the 3AS but also with the 4AS probes. Northern blotting with probes located upstream the 4AS probe gave the same pattern, indicating that the sequence of the 4.5 kb BCRF1 transcript is collinear to the genomic sequence (data not shown).

To further characterize the 5' end of the large transcript, S1 mapping, was performed as previously described by Giot et al. (1991), with a 5' a~P-labelled probe (AS 6141 to 5752) across the predicted 5' end of the 4'5 kb BCRF1 transcript. The results are shown in Fig. 2 (a): a strongly protected fragment of 280 bases, restricted to the anti-IgG-treated RNA, is consistent with a 5' end near 5860 (Fig. 2b). A sequence (TATTTA) about 36 bp upstream of this point, could serve as a TATA box (represented in Fig. 2b). All the results presented here (the length of the RNA, Northern blotting and S1 mapping) indicated that the 4.5kb BCRF1 transcript is collinear to the genomic sequence (5860 to 10280). A weakly protected band of 350 bases (Fig. 2a) was also observed, possibly corresponding to a small proportion of transcripts which were initiated 5' of the boundary mapped at 5860. The full-length protected band (Fig. 2a) may correspond to transcripts covering both BNRF1 and downstream sequence (represented in Fig. 2b) previously described by Hudson et al. (1985).

The induction time course of the BCRF1 transcripts was determined in anti-IgG-treated Akata cells, by Northern blot hybridized with the IAS ssDNA probe. As shown in Fig. 3 (a), the four transcripts were detected

Expression o f viral IL-IO in EBV-in fec ted cells 1167

at 9 h post-induction. The maximum yield of the three larger BCRF 1 transcripts was reached 9 h after treatment and the level remained unchanged over the next 15 h. The 0.8 kb transcript was detected at 6 h post-induction. The maximum yield of this transcript was also reached 9 h post-treatment, was unchanged for the next 3 h, strongly decreased 3 h later and remained unchanged from 15 to 24 h post-treatment.

Immunoprecipitation was performed on the super- natants of the anti-IgG-treated Akata cells. Samples corresponding to 2 x 10 ~ cells were removed at different times after induction (h) and supernatants were immuno- precipitated with a rabbit anti-vlL-10 polyclonal serum (499) raised in our laboratory. Immunoprecipitated complexes were loaded onto 15 % SDS-PAGE, Western blotted, probed with the same 499 serum and revealed with 125I-labelled Protein A. The results are shown in Fig. 3 (b). vIL-10 was detected 9 h after induction of the lytic cycle. The maximal yield was reached 12 h post-induction and the level remained unchanged from 12 to 24 h after induction of the lytic cycle.

We isolated four 3' co-terminal polyadenylated cDNAs containing the B C R F 1 0 R F from a cDNA library constructed with polyadenylated RNA extracted from TPA-treated B95-8 cells. The size of the four cDNAs indicated that all of them correspond to copies of mRNA initiated upstream of the putative BCR1 pro- moter. Thus, none could be compatible with copies of the 0"8 kb BCRF1 message, although Northern blotting indicates that the 0.8 kb transcript is abundant in TPA- treated cells. Interestingly, Lau et aL (1993) did not detect either any cDNA from oral hairy leukoplakia (OHL) corresponding to the 0.8 kb mRNA. The F1-3 cDNAs could correspond to copies of the 1.6 kb message which was determined to be unspliced by Lau et al. (1993), contradicting the results of Hudson et al. (1985). The sequence and the structure of the F4 cDNA shows that, transcript(s) are initiated upstream of the 1"6 kb message. Thus, our results confirm the inference, made from Sl mapping, of the existence of uncharacterized additional mRNA(s) initiated upstream of 8708 (Lau et al. 1993).

Northern blot analysis performed with RNA prepared from anti-IgG-treated Akata cells, showed that at least four late transcripts bear the BCRF1 open reading frame. In addition to the already described 0"8 and 1.6 kb bands, two other bands migrating at 3'5 and 4.5 kb could be clearly observed in anti-IgG-treated Akata cells. These bands were less visible in Akata and B95-8 TPA- treated cells (data not shown). The fact that Stewart et al. (1994) did not observe transcripts larger than 1.6 kb in Akata cells, may be due to the differences of treatment used to induce the lytic cycle. Similarly, in our experi- ments the 1 kb transcript is less important which may be

due to the difference in inducing agent of the lyric cycle. The large BCRF1 transcripts of 3-5 and 4"5 kb could explain the existence of F4 cDNA isolated from B95-8 cDNA library.

Northern blotting performed with probes covering regions located either upstream or downstream of the FR indicated, surprisingly, that within the 3"5kb transcript, a splicing event might occur in this region. Experiments using rapid amplification of 5' cDNA ends allowed us to isolate clones in which the FR sequence was 'scrambled' (data not shown). This may be due to the structure of the m R N A within the FR which may contain a strong stop signal for the reverse transcriptase. The 4"5 kb transcript which is collinear with the EBV genome contained the whole FR region. No other transcripts have been described in this region. It seemed to be initiated just upstream of the polyadenylation signal of the BNRF1 m RN A and also overlapped the EBERs encoding region.

The time course expression of the 0-8 kb transcript differs from the larger ones since its production peaked at 9 12 h post-anti-IgG treatment followed by a gradual decrease in the amount of the 0.8 kb message. The level of the other 3 mRNAs remained stable as of 9 h post- induction of the lytic cycle.

Although the 0"8 kb transcript was visible as early as 6 h post-induction, the vlL-10 protein was only detected at 9 h after addition of anti-IgG. The vlL-10 protein was therefore detected when the large transcripts were observed. This might indicate that at least one of the large mRNAs could be translated. A possible alternative could be that the detection of protein could be due to the accumulation of translated product of the 0"8 kb mess- age. The state level of vlL-10 production in the culture medium was not further explored. This cytokine pro- duced by EBV-infected cells, might play a role in the EBV-host relationship during the lytic phase of its life cycle.

We thank P. Y. Bourillot for his help in performing S1 mapping and D. Martel-Renoir for her participation in cDNA library construction. We are grateful to A. Sergeant for careful reading of the manuscript and M. Lee for proof reading. This investigation was supported by grants from 'la Ligue Nationale contre le Cancer' and the 'Institut Gustave Roussy' (CRC no. 94.7). R. Touitou was a recipient of grants from MRT and ARC.

References BAER, R., BANKIER, A. T., BIGGIN, M. D., DEININGER, P. L., FARRELL,

P. J., GIBSON, T. J., HATFULL, G., HUDSON, G. S., SATCHWELL, S. C., SEGUIN, C., TUFFNELL, P. S. & BARRELL, B. G. (1984). DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, 207-211.

GIOT, J.-F., MIKAELIAN, I., BUISSON, M., MANET, E., JOAB, [., NICOLAS, J.-C. & SERGEANT, A. (1991). Transcriptional interference between the EBV transcription factors EB1 and R: both DNA-binding and activation domains of EB1 are required. Nucleic Acids Research 19, 1251-1258.

1168 R. Touitou, C. Cochet and I. Joab

HUDSON, G. S., BANKIER, A. T., SATCHWELL, S. C. & BARRELL, B. G. (1985). The short unique region of the B95-8 Epstein-Barr virus genome. Virology 147, 81-98.

LAU, R., MIDDELDORP, J. & FARELL, P. J. (1993). Epstein-Barr virus gene expression in oral hairy leukoplakia. Virology 195, 463-474.

LOTZ, M., TSOUKAS, C.D., FONG, S., CARSON, D.A. & VAUGHAN, J. H. (1985). Regulation of Epstein-Barr virus infection by recombi- nant interferons selected sensitivity to interferon-y. European Journal of Immunology 15, 520-525.

MOORE, K. W., O'GARRA, A., DE WAAL MALEFYT, R., VIE1RA, P. & MOSMANN, T.R. (1993). Interleukin-10. Annual Review of Im- munology 11, 165-90.

ROUSSET, F., GARCIA, E., DEFRANCE, T., PERONNE, C., VEZZIO, N., HSU, D.H., KASTELEIN, R., MOOr.E, K.W. & BANCHERAU, J. (1992). Interleukin 10 is a potent growth and differentiation factor for activated human B lymphocytes. Proceedings of the National Academy of Sciences, USA 89, 1890-1893.

STEWART, J. P., BEHM, F. G., ARRAND, J. R. & ROONEY, C. M. (1994). Differential expression of viral and human interleukin-l0 (IL-10) by primary B cell tumors and B cell lines. Virology 200, 724-732.

TAKADA, K. & ONO, Y. (1989). Synchronous and sequential activation of latently infected Epstein-Barr virus genomes. Journal of Virology 63, 445M49.

VIEmA, P., DE WAAL-MALErYT, R., DANG, M. N., JOHNSON, K. E., KASTELEIN, R., FIORENTINO, D. F., DEVR1ES, J. E., RONCAROLO, M. G., MOSMANN, T.R. & MOORE, K.W. (1991). Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones : homology to Epstein-Barr virus open reading frame BCRFI. Proceedings of the National Academy of Sciences, USA 88, 1172-1176.

YATES, J. L., WARREN, N., REISMAN, D. & SUGDEN, B. (1984). A cis- acting element from the Epstein-Barr virus genome that permits stable replication of recombinant plasmids in latently infected cells. Proceedings of the National Academy of Sciences, USA 81, 3806-3810.

(Received 26 October 1995; Accepted 12 January 1996)