Molecular mimicry of the antigen receptor signalling motif by transmembrane proteins of the Epstein-Barr virus and the bovine leukaemia virus

Molecular mimicry by transmembrane

Gottfried

Alber,

of the antigen receptor

signalling motif proteins of the Epstein-Barr virus and

the bovine

leukaemia

Kwang-Myong

Kim, Peter Weiser,

Rita Carsetti Max-Planck-lnstitut

fur

and Michael

Immunbiologie,

Background: Many transmembrane proteins of eukatyotic cells have only a short cytoplasmic tail of 10 - 100 amino acids, which has no obvious catalytic function. These tails are thought to be involved either in signal transduction or in the association of transmembrane proteins with the cytoskeleton. We have previously identilled, in the cytoplasmic tails of components of B and T lymphocyte antigen receptors, an amino-acid motif that is required for signalling. The same motif is also found in the cytoplasmic tails of two viral proteins: the latent membrane protein, LMP2A, of Epstein-Barr virus and the envelope protein, gp30, of bovine leukaemia virus. Interestingly, both viruses can activate infected B lymphocytes to proliferate, as does signalling by the B-cell

receptor. Current

virus

Biology

Sttibeweg

Christa

Reth

51, W-7800

Freiburg,

Germany

Results: In this study, we show that the cytoplasmic tails of the two viral proteins, and the cytoplasmic tail of the B-cell receptor immunoglobulin-cl chain, when linked to CD8 in chimeric transmembrane proteins, can transduce signals in B cells. Cross-linking of these chimeric receptors activates B-cell protein tyrosine kinases and results in calcium mobilization. Furthermore, these cytoplasmic sequences are also protein tyrosine kinase substrates and may interact with cytosok proteins carrying SH2 protein-protein interaction domains. Conclusion: Our lindings suggest that viral transmembrane proteins can mimic the antigen-induced stimula tion of the B-cell antigen receptor and thus can intluence the activation and/or survival of infected B lymphocytes. 1993, 3:333-339

Background

Consensus

Immune recognition receptors such as the B-cell antigen receptor (BCR), the T-cell antigen receptor (TCR), and the Fc receptors for immunoglobulins IgG and IgE,

are multimeric protein complexes consisting of ligandbinding chains and subunits that are involved in intracellular signalling (for review see [ 1 ] ). Although ligand binding requires specific receptor sequences in each case, signal transduction appears to require a common amino-acid motif [ 2-4, the YXXL motif, which is conserved between BCR-Ig-a, BCR-Ig-P, TCR-5, CD3-r, 6, E, FcyRIWy and FcsRI-P and y chains [ 51, In a computer search ‘of a protein data bank for this signailing motif (D/E-X7-D/E-X2-Y-X2-L-X7-Y-X24/L), we have previously found [5] that this conserved aminoacid sequence is present in the cytoplasmic tail of the envelope protein gp30 of bovine leukaemia virus (BLV) [6]. As the precise position of the negatively charged amino acids in the motif is not conserved in all receptors (Fig. 1) we also conducted a search with a part of the motif containing only the conserved tyrosine and leucine/isoleucine residues. In this search, we found another viral transmembrane protein that has the YXXL motif in its cytoplasmic tail, namely the latent membrane protein 2A (LMWA) of Epstein-Barr virus (EBV) [7] (Fig. 1, line 13; also called terminal protein 1 [S]). EBV is a human herpesvirus that infects human B lymphocytes and eflkiently immortalizes them for Correspondence

to: Michael

Riesterer,

(1)

CD3CD3- 8 CD3-E TCR-6 TCRTCR- 5

(2) (3) (4)

121

1 2 3

(7)

FceRI-y FceRIj3

(9) (10)

BClvlg-a

(11) (1a (13)

BLV-gp30-1 BLV-gp30-2 EBV-LMPZA

(14)

y2a

(8)

BCWlg-j3

DRHSD

YQPL

GTQDQSL

YLGL

0 19‘33 Current Biology

Fig. 1. Occurrence

of the conserved YXXL motif in the cytoplasmic domains of the signalling subunits of the murine T-cell receptor CTCR) (lines lib), FcERI (lines 7, 8), B-cell receptor (BCR) (lines 9, IO), and the viral transmembrane proteins BLV-gp30 (lines 11, 12) and EBV-LMPZA (line 13). An unrelated Tyr motif in the carboxyl terminus of the membrane-bound y2a chain is shown for comparison (line 14). The sequences are aligned according to conserved Tyr and Leu/lle residues (red). * marks the position of the negatively charged amino acid. Gaps are indicated by l .

in vitro growth

[9]. It is associated with infectious

Reth

@ Current

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1993

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3 No

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-

mononucleosis, nasopharyngeal carcinoma, African Burkitt’s lymphoma and lymphoproliferative disorders that occur in immunosuppressed individuals. LMP2A is a multi-barrel transmembrane protein that crosses the lipid bilayer 12 times and has an amino-terminal cytoplasmic tail of 12i! amino acids (Fig. 2b). Recently, LMP2A has been shown to be associated in the cytoplasm with a 70kD protein and with Src family protein kinases [ 10,111. It was also found to be a tyrosine kinase substrate in EBV-transformed B lymphocytes [ 101. However, its function within EBV-infected B lymphocytes is unclear. (a)

Bovine leukaemia virus

(b)

Epstein-Barr

receptor-binding domain; gp30 is the transmembrane subunit and has a carboxy-terminal cytoplasmic tail of 58 amino acids (Fig. 2a). Recently, a new method has been developed that uses chimeric transmembrane molecules to test the signailing function of cytoplasmic sequences. Chimeric molecules made of CD8, CD4 or Tat, carrying the cytoplasmic tail of either TCR-6, CD3-s, FcsRI-y or BCR-Ig-a have thus been shown to transduce signals upon crosslinking [ 13-171. This signalling function is dependent on the presence of the conserved YXXL motif in the cytoplasmic tails [ 3,4].

virus

Results

To test the signalling capacity of the viral proteins, we constructed vectors that express chimeric molecules in which the extracellular and transmembrane portions of CDS are fused with the cytoplasmic tail of either LMP2A or gp30 (Fig. 2~). As controls, we used vectors expressing CD8 chimeric proteins with the cytoplasmic tail of either the Ig-a protein or the membrane-bound IgG2a heavy chain (y2a). The y2a sequence contains a tyrosine residue but does not have the YXXL motif (Fig. 1, line 14).

8PSl

(a) (c)

B-cell

receptor

and CD8

chimeric

molecules

cellS

“IL

Number

CDtWa

of

Cd ‘

>

‘,

~ttXMg-a

,,

loo1

II

BLV is a retrovirus that is most closely related to human T-cell leukaemia virus [ 121. It is the aetiological agent of bovine leukosis, a lymphoproliferative disease that is associated with tumlours of the B-cell lineage after a long latency period. H[ow such tumours are induced is not known. The BLV envelope glycoprotein complex, gp51/gp30, plays a critical role in the infection and syn cytium induction of B cells, as has been demonstrated using monoclonal ant&p51 antibodies [ 121. The gp51 protein is the extracelluiar subunit and contains the cell

(d) ] h

CD8/8p30

of

IO0

Fig. 2. Supposed structure of (a) the BLV envelope glycoprotein complex gp51/gp30 and (b) the EBV protein LMP2A. (c) Model of the IgG B-cell antigen recleptor and of the CD8 chimeric transmembrane proteins expressed on the surface of the transfected K46 B lymphoma cells. Due to the dimerization function of CD8, the chimeric molecules are expressed as homodimers.

CD8/LMPZA

celIs oL ‘B Number

B 1993Cuirent Biology

(b) kg-2a

IO’

IO2

lo3 IO4

anti-CD8a

IO0 IO’

lo2

IO3

IO4

(FITC)

J

Fig. 3. Detection of CD8cr on the surface of K46 cells transfected with expression vectors for the chimeric CD8a molecules: (a) CD8/y2a, (b) CD8/lg-a , fc) CD8ILMP2A and (d) CD8igp30. The parental and transfected cell lines were analysed by flow cytometry (104 cells per histogram) after staining with a fluorescein-labelled monoclonal rat anti-mouse CD8a antibody (Serva).

The different expression vectors were stably transfected into the IgG2a-producing B-lymphoma cell line K46 [ 181. For further experiments, we chose the CDSpositive ~46 transfectants K46-CD8/y2a, K46CD8/Iga, K46--CD8/IMP& and K46CD8/gp30 (Fig. 3a-d) which expressed homodimers of the chimeric CD8 molecules on their surfaces (Fig. 2~). For each chime&

,‘viral mimicry

of the antigen

receptor

Alber

et al.

RESEARCH

construct, three to four independent transfectants have been studied in tlhe functional assays described below. The responses obtained from independent transfectants were very similar. Signal transduction by the BCR involves the activation of protein tyrosine kinases (PTKs), which rapidly phosphorylate several substrate proteins [ W-211. We first tested the substrate phosphorylation induced by IgG2a-BCR in the four different CD8chimeric transfectants of K46. Crosslinking of the BCR with antiIgG antibodies resulted in tyrosine phosphorylation of substrate proteins ~158, ~127, ~115, ~103, ~82, ~70, ~65, p55 and p43 (Fig.4 lanes 3,6,9,12). The identity of most of these substrates is at present unknown, except for p43 which has been identified as mitogen-activated protein (MAP2) kinase [ 22-241. Cross-linking by anti-CD8 antibodies of the CD@al pha , CDQ’gp30 and CD8/LMP2A chimeric molecules expresssed on transfected ~46 cells also results in increased protein tyrosine phosphorylation (Fig. 4, lane 5,8,11). Although these responses are weaker than those induced by the BCR, they result in tyrosine

CD8Iy2a

phosphotylation of similar, or identical, substrate proteins, including the 43 kD MAP2 kinase. No increase in tyrosine phosphorylation was detected in the ~46 transfectant upon cross- linking the CD8/y2a molecule (Fig. 4, lane 2). The YXXL motif seems to have a dual role in signal transduction. It is involved not only in the activation of PTK, but also serves as a substrate of these enzymes. Indeed, by a mutation analysis of the cytoplasmic Ig-a sequence we, have shown that of the four tyrosines present, only the two in the YXXL motif become phosphotylated after receptor cross-linking (Flaswinkel H, Reth M, unpublished observations). To test the potential of tyrosines in the cytoplasmic tail to act as PTK substrates, we have used fusion proteins between glutathione-S-transferase (GST) and the cytoplasmic sequences of the BCR components y2a, Ig-a, Ig-p, and the EBV transmembrane proteins LMP2A and LMPl, in an in vitro kinase assay using the Src-related tyrosine kinase Fyn. As shown in Figure 5, only GST fusion proteins carrying a YXXL motif - Ig-a, Ig-p and LMP2A - become phosphorylated in this assay. By

cDWgp30

awIg-cl

PAPER

CDULMP2A

-1-m-

200

-

97.4

-

69

-

46

21.5 1

2

3

4

5

6

7

8

9

10

11

12

Fig. 4. Tyrosine phosphorylation of proteins after stimulation of K46 cells expressing CD8/y2a tail (lanes l-3), CD8ilgcc tail (lanes 4-61, CDEVgp30 tail (lanes 7-9), and CD8/LMPZA tail (lanes 10-12). Phosphoproteins of unstimulated cells (lanes 1,4,7,10), of cells stimulated with anti-CD8 antibodies (lanes 2,5,8,11) or with anti-l& antibodies (lanes 3,6,9,12) are shown. The relative positions of the molecular size standards are indicated in kilodaltons on the left.

335

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contrast, the cytoplasmic sequences of y2a and LMPl are not PTK substrates: both of these have a tyrosine at the carboxyl terminus but do not contain a YXXL motif. The substrate function of GST/gp30 could not be tested in this assay as no stable fusion proteins were produced by the bacteria that were transfected with the corresponding expression vector.

responses of the K46 transfectants expressing tl-r$ viral tail proteins were similar to that of the ac$ vated K&-CDS&a cells, although the kinetics of t&j calcium increase in K46CD8/LMP2A cells were slow& than in K4&CD8/gp30 cells (Fig. 6b). NO calcium re; sponse was detected after cross-linking of CD~/LMP~ molecules on a B lymphoma cell line (data not shown):

400 69

-

46

-

(b) I

anti-CST

200

I

0 69

l

I

I

I

I

I

I

60

120

180

240

300

360

Time

(set)

I

420

480

: -

469 -

30

l

.’ ”

Fig. 6. Changes in intracellular free calcium monitored in K4f transfectants after stimulation with anti-CD8 or anti-IgC antibod, ies. (a) Anti-CD8 antibodies were used to cross-link the CD8iy2: tail (0) and the CD8/lg-a tail (A), and anti-l& antibodies were used to cross-link membrane IgG on the K46 CD8/y2a transfectant (0). The anti-IgC-induced response in K46 CD8/y2a transfectants was comparable to the response in the other transfectants. (b) Anti-CD8 antibodies were used to cross-link the CDB/LMP2A tail (A) and the CD8igp30 tail (0).

anti-PY ‘.

Discussion Fig. 5. In vitro kinase assay of CST fusion proteins carrying the cytoplasmic sequence of y.2a, Ig-a, Ig-p, LMPZA or LMPI. After 10 % SDS-PAGE, the fusion proteins were detected on the same Western blot by either a monoclonal anti&ST antibody or the anti-phosphotyrosine antibody 4GlO (anti-PI’).

In response to cross-linking of the BCR, B lymphocytes mobilize calcium ions [25,26]. An increase in intracellular free calcium is detected in K46 cells upon exposure to anti-IgG antibodies (Fig. 6a). Cross-linking of the CD~ chimeric molecules on the K46 transfectants also results in a calcium response in K&CD8/Ig-a but not in K46CD8/y2a cells (Fig. 6a). The calcium

Our data show that the cytoplasmic tails of the viral proteins gp30 and LMP2A can transduce similar signals to the response triggered by the cytoplasmic tail of Ig-a, whereas the cytoplasmic tails of IMP1 and y2a, which lack the YXXL motif, have either a different signalling function or no signalling function at all. The YXXL motif, which is part of the viral proteins, seems to interact with the tyrosine kinases implicated in BCR signalling. Indeed, we have shown that the YXXL motif, which is often close to negatively charged amino acids (Fig. 1) is an efficient substrate for a tyrosine kinase of the Src family. After phosphorylation

Viral

mimicry

of the antken

receDtor

Alber

et al.

of its tyrosine residues, the YXXL motif may interact with proteins with SH2 domains that recognize phosphotyrosine within this context. The duplication and conserved spacing of the YXXL motif in the viral cytoplasmic tails suggest that proteins with two SH2 domains may bind to these tails. Indeed, B cells express a number of proteins known to have two SH2 domains each, namely phospholipase C-y, GTPase-activating protein, phosphotyrosine phosphatase PTPlC [ 271, and the recently identified tyrosine kinase Syk [28] that is presumed to be the homologue of the mouse PTK72 [ 291. The model outlined above is supported by data showing that both the TCR 5 subunit [30] and LMP2A in EBV-infected B cells [lo] are phosphorylated on ty rosine and are associated with a 7OkD tyrosine kinase with characteristics similar to those of syk. Although BLV and EBV have rather different genomic organizations, they are similar in that they both cause a strong proliferative response, in infected bovine and human B lymphocytes, respectively. This B-cell proliferation seems to be due, in part, to the expression of nuclear factors encoded by these viruses [31,32]. The expression or activation of these factors may, however, also be controlled by external signals. Our findings that the cytoplasmic sequence of gp30 and LMP2A can generate signals that mimic those induced by the BCR - suggest that the viral proteins can signal in infected B cells. How, and at what stage dluring the virus life cycle, B-cell signals are induced by the viral proteins is not yet clear. The LMP2A gene has recently been eliminated from the EBV genome by targetted mutation [33,34]. The mutated, LR/IP2Anegative, virus was still able to grow and transform human Es cells in vitro. This suggests that LMP2A is not required for growth in vitro but plays a role in the propagation of the virus in vivo. Activation of the BCR is required in vivo for the generation of memory B cells; signals from LMP2A and, presumably, gp30 may allow infected B cells to enter the pool of long-lived memory B cells in an antigenindependent manner, thus explaining the state of latency observed with these viruses. Indeed, LMP2A is the only EBV protein expressed in latently infected B cells [35,361.

Conclusion

Our data suggest that EBV and BLV, two very different viruses both of which infect B cells, have evolved similar mechanisms of interaction with the signalling machinery in these cells. Both express transmembrane proteins that carry in their cytoplasmic tail a YXXL motif; this motif plays a crucial role in signalling from the BCR. The signalling mimicry of EBV and BLV may be part of a general viral strategy to optimize multiplication. Indeed, many other viruses can induce signals in infected cells. Some carry part of a cytokine

RESEARCH

PAPER

receptor gene in their genome [ 371; others produce viral proteins that bind to, and activate, cellular receptors [38]. Materials and methods Vector construction For the construction of chime&

CD8 molecules, the DNA encoding the cytoplasmic portions of y2a (residues 648-674; [39]), Ig-a (residues 162-220), BLV-gp30 (residues 153-213) and EBV-LMP2A (residues19122) was amplified by the poly merase chain reaction (PCR) using a S’oligonucleotide and a 3’oligonucleotide with a BarnHI and SmaT restriction site, respectively. The fragments were sequenced using the Sanger dideoxynucleotide technique and cloned into BumHI/SmuL linearized CD8 vector. The CDS expression vector pLVLy2-Hy is a derivative of pUC19; it carries the long terminal repeat (LTR) promoter of the Moloney murine leukaemia virus, and part of the Lyt2 cDNA from the NcoI site at the AUG start codon down to an introduced BumHI site at a position three codons (CAC AGG ATC C) after the end of the transmembrane sequence. Thus, the chimeric protein carries only three cytoplasmic amino acids of the wild-type CD8 and does not contain the Ick binding site [40]. The BamHI site is followed by a unique SmaI site and a sequence carrying the intron and the polyA site of the small t antigen of SV40, and the hygromycin resistance gene of the pHMR272 vector [41]. ~46 cells were stably transfected as described previously [42] and the K46CD8/y2a, K46CD8/gp30, and K46CD8/LMP2A cells were selected in medium containing 750 U/ml hygromycin (Calbiochem). Expression of chimeric molecules was tested on a FACScan after staining cells with a fluoresceinlabelled monoclonal rat anti-mouse CD8a antibody (TD3 105). Detection of Tyrosine Phosphorylation Cells were stimulated for 1Omin at 37°C by monoclonal rat anti- mouse CD8a (TIB105; 2Opg/ml) or goat anti-mouse IgG (Sigma; 10 &ml) before solubilization in buffer containing 1% Triton X-100 and protease and phosphatase inhibitors. Unstimulated cells were used as controls for basal cellular tyrosine phosphorylation. SDS/2-mercaptoethanol extracts were electrophoresed on a 10 % SDS polyacrylamide gel (200,000 cell equivalents/lane) and transferred to nitrocellulose. The membrane was incubated with 0.2 l&ml of monoclonal anti-phosphotyrosine antibody (4GlO; Upstate Biotechnology Inc.) and stained with 0.4 pg/ml of horseradish peroxidase-conjugated goat anti-mouse x(Southern Biotechnology Associates Inc.). Tyrosine-phosphorylated proteins were detected by chemiluminescence (ECL kit; Amersham) according to the manufacturer’s directions. In vitro kinase assay Expression of the different GST fusion proteins in the transfected bacteria growing in logarithmic phase was induced with IPTG (Sigma, St. Louis, MO). After 2 h incubation, bacteria were pelleted and sonnicated. Fusion proteins were purified with glutathione-conjugated Sepharose (Pharmacia, Freiburg, FRG). Aliquots of fusion protein bound to Sepharose beads were washed once in kinase buffer (50 mM Tris-HCl pH 7.6 and 1OmM MnCl). Baculovirus-expressed human Fyn extract (gift from Sara Courtneige) was diluted I:20 in kinase buffer containing 1 mM ATP and added to the Sepharose beads. Following an incubation of 3Omin at 3O”C, the beads were washed once with PBS and then boiled in SDS sample buffer. Proteins were size separated by SDS-PAGE and blotted onto nitrocellulose.

337

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1993, Voi 3 No 6

Phosphotylated cyrosine antibody,

Measurement

proteins 4G10,

of

calcium

were using

detected by the the ECL detection

anti-phosphosystem.

release

15.

16.

Cells were loaded in Iscove’s medium containing 1% inactivated fetal calf serum, 2 pM indo-l acetoxymethylester (Molecular Probes), and 0.02 % (WV/V) pluronic F-127 (Molecular probes) for 45 min at 37°C in the dark [43]. Subsequently,measurements were carried out at 37°C in a FACStar Plus cell sorter (Becton

Dickinson). After 70 set, 25 &ml of the monoclonal rat antimouse anti-CD8a antibody (TIEUOS)or goat anti-mouse IgG antibody (Sigma) was added, and the mean fluorescence ratio of calcium-bound to calcium-free indo-l (‘FL4 ratio’, a measure of relative calcium concentration) recorded. Tracings were

17.

18.

122:54%554. 19.

monitored for 7 min using the CHRONYSprogram. Acknowledgements We thank Drs Yoji Ikawa, G. Iaux, N. MuellerLantzsch and K. Eichmann for providing plasmids with the envgp30 sequence, pSP64TP1, pLMP and plyt-2a, respectively, and Sara Courtneidge for the m extract. We also acknowledge the expert technical assistance of H. Kohler in the calcium measurement. GA. is a recipient of a Habilitation Stipend fellowship from the Deutsche Forschungsgemeinschaft and K.-M.K. is supported by an Alexandervon-Humboldt fellowship.

20.

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3.

4.

5.

BEYER~ AD, SPRWT tions of the T-cell

LL, W~LUAMS AF: MuItimolecular associaantigen receptor. Trends Cell Biol 1992,

Nature

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LI JP, D’ANDREA AD, LODISH HF, BALTIMORE D: Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor. Nature 1990, 343~762-764. KABAT EA, Wu m, PERRY HM, Go’rrmm KS, FOELLER C (Em). In Sequences of proteins of immunological interest, Washington: NIH Press; 1991, 1:713.

RESEARCH

40.

41.

42.

43.

PAPER

TAGAWA M, NAKAUCHI H, HERZENBERG IA, NOLAN GP: Formal proof that Merent-size Lyt-2 polypeptides arise from Merential splicing and post-transcriptional regulation. Proc Nat1 Acud Sci USA 1986, 83~3422-3426. BERNARD HU, KRAMMER G, R&mm@ WG: Construction of a fusion gene that confers resistance against hygromycin B to mammalian cells in culture. Exp Cell Res 1985, 158:237-243. HO~IBACH J, LECLERCQ L, RADBRUCH A, R&JEWSKV K, RETH M: A novel 34kD protein co-isolated with the IgM molecule in surface &M-expressing cells. EMBO J 1988, ~3451-3456. OWEN CS: Quantitation of lymphocyte intracellular free calcium signals using indo-1. Cell Calcium 1988, 9~141-147.

Received: Accepted:

Apd llb4ay

8

1993; revised: 1993.

7 h@’

19%.

339