Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Schloßgarten 4, D-91054 Erlangen, Germany1
Institute for Radiobiology at the Paul Scherrer Institute, CH-5232 Villigen-PSI, Switzerland2
Author for correspondence: Brigitte Biesinger. Present address: Department of Molecular Biology, Max-Planck-Institute for Biochemistry, Am Klopferspitz 18A, D-82152 Martinsried, Germany. Fax +49 89 8578 2454. e-mail biesing{at}biochem.mpg.de
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Abstract |
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In order to approach the transforming functions of HVS subgroup B, we first analysed the left-terminal L-DNA of strain SMHI. Virion DNA was cloned into pBluescribe M13+ and plasmids containing left-terminal sequences were identified by colony hybridization with a radioactively labelled fragment of strain C-488 (nt 26836652). Four overlapping PstI and NsiI fragments were sequenced as described previously (Albrecht et al., 1992 ; Ensser et al., 1997
) and analysed with the GCG programs using standard parameters (version 9.0; Genetics Computer Group, Madison, WI, USA). The nucleotide sequence of this region was collinear with the left terminus of the HVS A-11 genome, with an overall identity of 59%, ORFs and HVS U RNA (HSUR) genes at equivalent positions and a gap at nt 3130 (Fig. 1A
). The reading frames for dihydrofolate reductase, which was part of the hybridization probe, were well conserved among HVS strains A-11, B-SMHI and C-488 and the human gene (7985% amino acid identity for each individual pair). The putative HSUR transcripts (seven genes in HVS A-11 and B-SMHI; four genes in HVS C-488) showed 5084% nucleotide identity among the three virus isolates. However, conservation was significantly lower at the very left end of L-DNA encoding the stp genes of subgroups A and C. The polypeptide encoded by B-SMHI, by analogy designated StpB, showed little similarity to StpA (amino acid identity/similarity 28/35%) or StpC (22/29%). All Stp proteins shared a C-terminal hydrophobic region long enough to serve as a membrane anchor. The GX1X2 motifs (X1 and/or X2 is P) present in StpC form a collagen-like cluster of 18 tandem copies (Biesinger et al., 1990
) and StpA contains up to nine copies (Lee et al., 1997
). In contrast, we found only three non-clustered GX1X2 motifs in StpB, most likely reflecting random amino acid distribution. The TRAF-binding motif PXQXS/T (Devergne et al., 1996
) occurred once in StpA and twice in StpB (Fig. 1B
). Finally, the amino acid sequence YAEV of StpA is conserved in StpB as YAEI (Fig. 1B
). This YAEV/I motif resembles the consensus for phosphotyrosine-dependent binding to the SH2 domains of Src family tyrosine kinases (Songyang et al., 1993
). Acidic residues N-terminal of this motif that are characteristic for preferred Src kinase substrates (Songyang et al., 1995
) are present in StpA but not in StpB. In fact, StpA is phosphorylated on tyrosine residues in vivo in the presence of Src and the tyrosine residue of the YAEV motif has been shown to be essential for the interaction with cellular Src (Lee et al., 1997
), suggesting that StpA binds Src via the SH2 domain.
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The importance of the YAEI motif for binding suggested that association of StpB with Src is mediated by the SH2 domain. As SH2 binding is dependent on tyrosine phosphorylation, we analysed the phosphorylation state of AUStpB in vivo and in vitro after expression in COS cells (Fig. 3A, B
). AUStpA was included in this assay as a positive control. The viral proteins were expressed alone (Fig. 3A
, B
; lanes 2 and 3), together with c-Src (lanes 5 and 6) or with an inactive point mutant of c-Src to exclude adaptor effects (lanes 8 and 9). Expression of c-Src was monitored by EC10 immunoblot of the cell lysates (Fig. 3A
, middle panel) and indicated comparable levels of wild-type (lanes 46) and mutant (lanes 79) proteins. The AU1-specific immunoblot (Fig. 3A
, lower panel) showed expression of StpA and StpB in the corresponding lysates. Reduced expression levels after co-expression of Src most likely reflect the limitations of the overexpression system used. StpA migrated as a doublet of approximately 24 and 32 kDa, but the slower form was detected only after very long exposure when inactive Src was co-expressed (lane 8 versus lanes 2 and 5; data not shown). StpB was present as a triplet and two additional, slower migrating forms appeared after co-transfection with c-Src (lane 6 versus lanes 3 and 9). Thus, the modifications of both StpA and StpB that lead to retarded migration appear to be regulated by the activity of Src. As all forms of these proteins may be phosphorylated on tyrosine residues (see below), we suggest that the observed modifications were induced by serine/threonine kinases acting as downstream effectors of Src.
The anti-phosphotyrosine immunoblot (Fig. 3A, upper panel) revealed a strong phosphorylation of all StpB species in vivo in the presence of c-Src (lane 6). The amount of phosphorylated StpA was significantly smaller (lane 5) and slower migrating forms were only visible after longer exposure (data not shown). Comparison of the phosphotyrosine-specific and epitope-specific signals (Fig. 3A
, lower panel) suggested that StpB is phosphorylated in vivo more efficiently than StpA. Endogenous kinases (lanes 2 and 3) and inactive c-Src (lanes 8 and 9) did not lead to detectable levels of StpA or StpB phosphorylation in vivo. Aliquots of the same cell lysates were used for AU1-specific immunoprecipitation followed by an in vitro kinase reaction (Lang et al., 1997
) (Fig. 3B
). After co-expression with c-Src, AUStpB and AUStpA were phosphorylated in vitro to a comparable extent (lanes 5 and 6). In vitro phosphorylation of StpB by endogenous kinases alone (lane 3) or after co-transfection with inactive c-Src (lane 9) was detectable only after longer exposure and was significantly weaker than for StpA (lanes 2 and 8). These data suggest that StpB is a good substrate for Src or a Src-activated tyrosine kinase in vivo, while StpA might be more efficiently phosphorylated in vitro.
To prove binding of StpA and StpB to the SH2 domain of Src definitively, we performed co-immunoprecipitation assays with Src deletion mutants (Dunant et al., 1996 ). Both AUStpA and AUStpB were co-transfected with wild-type c-Src and with deletion mutants lacking the SH3 or SH2 domain (Fig. 3C
, D
). Expression of the Src constructs was monitored by an immunoblot with cell lysates and SRC2 antiserum (Fig. 3C
, D
; middle panels). The AU1-specific lysate immunoblot revealed expression of recombinant StpA and StpB in the appropriate lanes (Fig. 3C
, D
; lower panels). Immunoprecipitation with SRC2 antibodies followed by an AU1-specific immunoblot demonstrated interaction of StpA and StpB with wild-type and SH3-deficient Src (Fig. 3C
, D
; upper panel). In agreement with published data (Moarefi et al., 1997
), the SH3-deletion mutant appeared to be more active than wild-type Src, resulting in enhanced mobility shift and binding of StpB (Fig. 3D
; lanes 6 and 7). This effect of SH3-deficient Src was less pronounced for StpA, where mainly the slower-migrating form was co-precipitated (Fig. 3C
; lanes 6 and 7). In contrast, deletion of the SH2 domain abolished Src binding of both StpA and StpB (Fig. 3C
, D
; upper panel).
Thus, after co-expression with c-Src in COS cells, we did not observe significant differences in the ability of StpA and StpB to interact with the SH2 domain of c-Src via the YAEV/I motif. While acidic residues typical of a Src kinase phosphorylation site are present only in StpA, in vivo tyrosine phosphorylation in this system was higher for StpB. An endogenous tyrosine kinase activated by c-Src overexpression might account for this discrepancy by phosphorylating StpB and generating Src SH2-binding sites. Different expression levels of such a kinase might also explain the contradictory results obtained by Jung and co-workers using 293T cells (Choi et al., 2000 ).
As our nucleotide sequence indicated, HVS B-SMHI is more closely related to HVS A-11 than to HVS C-488 and, in spite of low similarity, StpA and StpB share all interaction partners identified so far, while StpC shows unique properties. This potent oncoprotein binds Ras and activates MAP kinases (Jung & Desrosiers, 1995 ), interacts with TRAFs and activates their downstream target, NF-
B (Lee et al., 1999
), and, finally, it carries collagen-like repeats capable of inducing protein multimerization (Choi et al., 2000
). All these properties are required for transformation of rodent fibroblasts. In addition, lymphocyte transformation and lymphoma induction by HVS C-488 depend on the presence of another viral protein, Tip, which is co-transcribed with StpC and may influence T cell growth regulation by its interaction with the T cell-specific tyrosine kinase Lck (Biesinger et al., 1995
; Fickenscher et al., 1996
; Duboise et al., 1998
; Isakov & Biesinger, 2000
).
In contrast, the ability of StpA and StpB to interact with TRAFs does not result in NF-B activation (Lee et al., 1999
; Choi et al., 2000
). Despite differences in their tyrosine kinase substrate properties, both proteins appear to bind the SH2 domain of Src with similar efficiency (Fig. 3
). However, only StpA is able to transform rodent fibroblasts, although with a moderate phenotype (Jung et al., 1991
). In accordance with our data, mutation of the Src-interacting sequences is not required to generate a transforming StpB variant. The component missing in StpB may be self-oligomerization (Choi et al., 2000
). As supposed but not proven by Jung and co-workers, this function may also be provided by the collagen-like triplets scattered within the N-terminal half of StpA. While StpA has long been known to be required for the oncogenic phenotype of HVS A-11 (Murthy et al., 1989
) and induces lymphoma by itself (Kretschmer et al., 1996
), the functions of StpB and of the interaction motifs in lymphocytes remain to be analysed. As demonstrated clearly by the work of Longnecker and co-workers on the EpsteinBarr virus protein LMP2A (Longan & Longnecker, 2000
; and references therein), growth-altering effects of viral transformation-associated proteins may depend significantly on the cellular context.
Taken together, the low efficiency of transformation by subgroup B strains seems not to be due to a failure of StpB to bind cellular proteins known to interact with StpA. Comparative analyses of the effects of StpA and StpB in lymphocyte transformation by recombinant HVS might help in future to delineate the role of cellular interaction partners and the influence of additional viral factors on lymphoma induction by subgroup A and B isolates of HVS.
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Footnotes |
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References |
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Received 31 August 2000;
accepted 12 October 2000.