(Received for publication, September 27, 1994; and in revised form, December 15, 1994)
From the garten 4, D-91054
Erlangen, Federal Republic of Germany, Bristol-Myers
Squibb, Department of Molecular Biology, Signal Transduction
Laboratory, Princeton, New Jersey 08543, and
Subgroup C strains of Herpesvirus saimiri, a
leukemogenic virus of non-human primates, transform human T cells to
permanent growth in culture. These cells retain their antigen
specificity, and they are becoming widely used as a model for activated
human T cells. Though a variety of human cell types can be infected by H. saimiri, transformation appears to be specific for
CD4+ and CD8+ T cells. Our investigation of early signaling
events in H. saimiri-transformed T cells revealed a novel
40-kDa phosphoprotein complexed with the T cell-specific tyrosine
protein kinase p56. This protein, termed Tip
(tyrosine kinase interacting protein), is identified as a viral protein
encoded by the open reading frame 1 (ORF1). In the transformed cells
Tip is expressed together with the gene product of ORF2, the viral
oncoprotein StpC, which acts on epithelial cells. The H. saimiri genome has 75 ORFs, but only ORF1 and ORF2 are transcribed in
transformed human cells. Tip is phosphorylated on tyrosine in cell-free
systems containing Lck, indicating that the viral protein is a
substrate for this T cell-specific kinase. Alteration of T cell
signaling pathways by Tip may be the second event complementing the
action of StpC in a new mechanism of T cell transformation.
We have previously shown that certain strains of Herpesvirus
saimiri subgroup C, a tumor virus of non-human primates, transform
human T cells to permanent IL-2-dependent ()growth in
vitro(1) . Immortalized human T cells do not produce
infectious virus but harbor 30-60 H. saimiri genome
equivalents per diploid cell genome in the form of circular episomal
DNA(1) . The immortalized cells show a mature activated
phenotype, expressing the
/
T cell receptor (TCR), CD4 or
CD8, IL-2 receptor
-chains (CD25), and MHC-II
molecules(1) . These molecules remain functionally competent as
demonstrated by activation of Src-related kinases, tyrosine
phosphorylation, and mobilization of
[Ca
]
(2, 3) .
The antigen-specific responses are also preserved by the transformation
process(3, 4, 5, 6) . It has been
shown that H. saimiri-transformed cells are hypersensitive to
CD2 ligation. Interaction of CD2 with LFA-3, which is also expressed on
these cells leads to secretion of IL-2 and to autocrine
growth(7) . After infection with H. saimiri the
resulting cell lines are remarkably stable. In more than 12 months of
continuous culture, they did not lose the TCR-CD3 complex or the CD4
coreceptor. Such stability is not seen in many human leukemic T cell
lines or in lines transformed by HTLV1, which tend to lose the TCR-CD3
complex sometimes after only 2-4 months of in vitro culture(8, 9) . Whereas many different cell types
can be infected persistently or in a productive cycle by H.
saimiri(10) , all immortalized T cells express a mature
phenotype with CD4 or CD8 on their surface. The reasons for the
selectivity of the transformation event have not been known. However,
taken together our observations have suggested that the signaling
apparatus of mature T cells may be required for the continuous
proliferation induced by H. saimiri.
Non-receptor protein
tyrosine kinases of the Src family play a crucial role in early
signaling in T cells. One of them, p56, is
expressed selectively in thymocytes, mature T cells, and NK-cells, and
it associates with the CD4 and CD8 coreceptors. After cross-linking of
CD4 or CD8 on the cell surface p56
becomes
enzymatically activated, which is followed by the tyrosine
phosphorylation of several substrates, among them the
-chain of
the TCR complex. T cells deficient in p56
fail
to respond to triggering of the TCR, indicating that this enzyme is
essential also for the antigen-specific response of T
cells(11, 12) .
The viral factors responsible for
the transforming effects of H. saimiri on T cells have not yet
been defined. Of the approximately 75 open reading frames (ORFs)
encoded by the viral genome (13) many have been tested for
transcription in H. saimiri-transformed T cells. So far, only
a single bicistronic mRNA from the left terminal DNA containing ORF1
and ORF2 (14) was regularly observed. ORF2 codes for the H.
saimiri-transforming protein StpC, which is translated in
transformed human T cells. ()Here we report that the ORF1
protein is also expressed in transformed human T cells where it
associates with the Src-related protein tyrosine kinase p56
and becomes phosphorylated on tyrosine. These findings
indicate that H. saimiri interferes with early signaling
events, and they provide an explanation for the T cell selectivity of
the growth transformation induced by H. saimiri. We therefore
suggest the name tyrosine kinase interacting protein (Tip) for the ORF1
gene product.
Nontransformed cells were cultured in complete medium, RPMI 1640
supplemented with 4 mML-glutamine, 0.015 M HEPES, 200 IU/ml penicillin, and 200 µg/ml streptomycin and
10% screened bovine or pooled human serum at 37 °C, 5% CO in a humidified atmosphere. After infection with H. saimiri cells were kept in 50% CG medium (Vitromex, Vilshofen) and 50%
complete medium supplemented with 40 units/ml human recombinant IL-2
(Boehringer Mannheim) or with 100 units/ml human recombinant IL-2
(Eurocetus, Amsterdam, The Netherlands).
Figure 3:
Characteristics of the H. saimiri ORF1 and Tip sequences. The panel shows the position of
ORF1 in the H. saimiri genome, indicates the length of the
bicistronic mRNA that is found in transformed T cells, and gives some
features of the ORF1-encoded protein H. saimiri-Tip. Potential
tyrosine phosphorylation sites (Y), a stretch homologous to
the SH3 binding site consensus sequence, and a stretch similar to the
Src family kinase regulatory domain are shown. The sequence of the
synthetic peptide used to generate the anti-Tip serum and the beginning
of the sequence expressed as fusion protein with -galactosidase
are indicated by arrows.
Amino-terminally truncated ORF1 (Fig. 3) and ORF2 as well as StpA coding sequences were fused to
-galactosidase by cloning into the procaryotic expression vector
pROS, and fusion proteins were purified as described(22) . A
fusion protein of p56
and glutathione S-transferase (GST-Lck) was produced in a baculovirus
expression system and purified by affinity chromatography on
glutathione-sepharose columns(23) .
Figure 1:
A
40-kDa phosphoprotein coprecipitates with p56 in
T cells transformed by H. saimiri but not in the noninfected
parental clones. T cells were lysed, and p56
was
precipitated with a polyclonal anti-Lck serum or coprecipitated using
the anti-CD4 mAb MAX.16H5 as indicated. After an immune complex kinase
reaction the proteins were separated by 8% SDS-PAGE. Molecular mass
markers are on the right; the positions of Lck and the 40-kDa
phosphoprotein are indicated on the left of the panel. The clones were 61/39 and 39-HVS (1), 61/48
and 48-HVS (2), 68/4 and 68/4-HVS (3), and 68/5 and
68/5 HVS (4). CB15 is a transformed T cell line derived from
cord blood T cells (see ``Experimental Procedures'' for a
description of these cell lines).
Figure 2:
Phosphorylation of the 40-kDa
phosphoprotein is increased after activation of
p56. 39-HVS cells were incubated with anti-CD4
mAb for 2 min followed by cross-linking with an anti-mouse serum for
the time indicated to activate p56
. After lysis
an immune complex kinase reaction was performed with enolase as an
exogenous substrate, and the proteins were separated by SDS-PAGE. The
phosphorylation of the 40-kDa band increases similarly to the substrate
enolase. The positions of Lck, enolase, and the 40-kDa protein are
indicated on the right.
Figure 4:
The 40-kDa phosphoprotein can be
reprecipitated from p56 immune complexes by
anti-Tip (ORF1) antibodies (A), and it is phosphorylated on
tyrosine (B). A, Lck was precipitated from CB23
cells, and an immune complex kinase assay was performed. After
disruption of the immune complexes proteins were reprecipitated using
different antisera as indicated. S. aureus alone served as
control. This was followed by SDS-PAGE and autoradiography. Molecular
standards are on the right, and the positions of Lck and the
40-kDa phosphoprotein are on the left. B, the 40-kDa
bands from the anti-Lck precipitate (left) and the anti-Tip
precipitate (right) were excised and subjected to phosphoamino
acid analysis. The position of phosphotyrosine is marked.
Reprecipitation with anti-phosphotyrosine antibodies (anti-PY)
confirmed the phosphotyrosine content of p40
(A).
Figure 5:
Reconstitution of p40 in uninfected T
cells by the ORF1/tip in vitro translation product. Uninfected
T cells (61/39) were lysed, and ORF1 in vitro translation
product (Tip) was added as indicated (+). After precipitation with
anti-Lck or anti-Tip and immune complex kinase reaction proteins were
separated by SDS-PAGE and autoradiographed. Molecular standards as well
as the positions of Lck and p40are shown.
S-metabolically labeled in vitro translated Tip
is shown for comparison.
Figure 6:
Lck phosphorylates the ORF1--Gal
fusion protein. An Lck immunoprecipitate from uninfected T cells was
added to
-Gal fusion proteins. Only the fusion protein containing
the ORF1 sequences (lane 3) was phosphorylated. StpA (lane
2) or StpC (lane 4) fusion proteins and
-galactosidase from the vector alone (lane 1) remained
negative.
Taken together, our data
show that the ORF1 gene product associates with and can be
phosphorylated by p56, and we propose to name it H.
saimiri tyrosine kinase interacting protein (Tip).
Our results show that the ORF1 gene product of H. saimiri C488, H. saimiri-Tip, is expressed in human T cells
transformed to permanent growth by the virus. The viral Tip is
associated with the protein tyrosine kinase p56 and can
be phosphorylated on tyrosine by this enzyme in cell free systems.
Activation of Lck after ligation of CD4 leads to an increase in
phosphorylation of Tip, supporting the idea that the viral protein is a
substrate for p56
. Interaction of Tip with the other
Src-like tyrosine kinases of T cells, p60
and
p62
, was not seen in the transformed human T cells. But
since these are expressed in T cells at much lower levels than Lck,
phosphorylation of Tip by the other enzymes might not reach the
threshold of detection even in a sensitive immune complex kinase assay.
Expression of Tip had not been described before, and the functions
of this viral protein are largely unknown. Some gene products of other
viruses have been reported to associate with tyrosine kinases. The
Epstein-Barr virus, another herpesvirus transforming human B
cells, expresses the latent membrane protein 2A (LMP2A), which has no
similarity to Tip except for the potential membrane anchoring. In the
transformed B cells LMP2A interacts with Src-related
kinases(28) , but it does not seem to be involved in the
transformation process itself (29) . LMP2A blocks the signaling
function of the surface immunoglobulins and thereby protects the cells
from lytical growth and prolongs latency of the virus(30) . A
second example is the bovine papillomavirus oncoprotein E5. It
associates with the receptor tyrosine kinases for platelet-derived
growth factor and epidermal growth factor and seems to constitutively
activate them by dimerization(31) . Finally, the middle T
antigen of polyoma virus links Src and related tyrosine kinases to PI3
kinase and to the Shc adapter molecule. Shc in turn binds Grb2, which
mediates activation of the Ras signaling cascade (32) .
However, Tip has no structural similarities to any of these proteins,
so analogies in function cannot be deduced.
A role for Tip in the
cell transformation process induced by H. saimiri has not been
shown before. Earlier studies had focused on the ORF2 gene product
StpC, which is encoded on the same mRNA. Deletion experiments revealed
that stp genes are necessary for monkey lymphocyte
transformation by H. saimiri subgroup A(33) . After
transfection into the rodent fibroblast cell line Rat-1, stp genes from H. samiri subgroups A and C caused focus
formation in vitro, whereas expression of the ORF1 gene
product (Tip) did not induce any morphological changes(34) .
The oncoprotein StpC induced numerous epithelial tumors in the founder
generation of transgenic mice, but their T cells appeared unaffected (35) . These findings attribute a strong transforming potential
to StpC in epithelial and fibroblast cells but not in murine T cells.
Accordingly, the effects of the isolated StpC observed in these rodent
systems cannot explain why wild type H. saimiri is able to
transform human T cells. Whereas various human cell types are infected
by the virus(10) , only mature human T cells are transformed to
continuous growth(1) . The selectivity of the viral
transformation is further underlined by the observation that numerous
attempts to transform immature thymocytes never resulted in CD4-/CD8-
double negative lines. ()In addition, the phenotype of H. saimiri-immortalized T cells is remarkably stable. Cell
surface markers of mature activated T cells, in particular the
/
TCR-CD3 complex, CD2, CD4 or CD8, the IL-2 receptor
-chain (CD25), and MHC-II molecules, have remained present and
functional in the cell lines even after more than 12 months of
continuous culture. There is also evidence for the involvement of CD2
in the continuous growth stimulation of T cells transformed by H.
saimiri(7) . These findings suggest that the mature
phenotype of T cells is a prerequisite for growth transformation.
Having demonstrated complex formation of H. saimiri Tip with
p56
we propose that H. saimiri subgroup C
utilizes the early T cell signaling pathways linked to the molecules
mentioned above. A potential mechanism for the action of H.
saimiri-Tip would be the alteration of the enzymatic activity of
p56
and/or other Src-related kinases. Alternatively,
similar to polyoma virus middle T antigen, Tip might serve as an
adapter linking p56
to other signaling molecules.
Alteration of the enzymatic activity of p56
, a tyrosine
kinase essential for signaling in T cells, could profoundly alter T
cell behavior. Expression of an activated form of Lck in a CD4-negative
T cell hybridoma augmented signaling through its TCR(36) .
Although it is not an obligatory kinase for IL-2 receptor signaling,
p56
is likely to participate in the IL-2 response. Lck
associates with the IL-2 receptor(37) , becomes transiently
activated after IL-2 binding(38) , and a decrease in CD4-bound
Lck activity reduces the T cell response to IL-2(39) .
Significantly, unphysiologically high levels of Lck activity are
oncogenic in transgenic mice (40) and lead to focus formation
by transfected fibroblasts(41) . Furthermore, there are
kinase-independent functions of p56
in T cell
signaling(42) , probably mediated by its SH2 and SH3 domains.
These might be influenced by Tip. Facilitation of complex formation
between Lck and one of its substrates might greatly enhance the
efficiency of a signaling pathway without changing the overall
enzymatic activity of p56
.
Both the StpC and the Tip equivalents of another H. saimiri subgroup C strain have been shown to be necessary for full initial activation of human T cells by the virus(43) . These short term experiments do not allow conclusions about the permanent growth transformation of human T cells by H. saimiri. However, they imply a cooperation of Tip and StpC in T cells, which is in accordance with the reports on transformation by the StpC and the data on Tip we present here. As a conclusion we suggest that H. saimiri expresses two factors in human T cells with StpC acting as the basic oncoprotein, which is complemented by the T cell-specific action of Tip. The cooperation of these two factors would not only explain the T cell selectivity of the viral tranformation process but also promises new insights into T cell activation and growth control.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) HSV 488A-M55264[GenBank].