©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Product of the Herpesvirus saimiri Open Reading Frame 1 (Tip) Interacts with T Cell-specific Kinase p56 in Transformed Cells (*)

(Received for publication, September 27, 1994; and in revised form, December 15, 1994)

Brigitte Biesinger Alexander Y. Tsygankov (1) Helmut Fickenscher Frank Emmrich (2) Bernhard Fleckenstein Joseph B. Bolen (1) Barbara M. Bröker (2)(§)

From the  (1)Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, Schlobetagarten 4, D-91054 Erlangen, Federal Republic of Germany, Bristol-Myers Squibb, Department of Molecular Biology, Signal Transduction Laboratory, Princeton, New Jersey 08543, and (2)Max-Planck-Gesellschaft, Arbeitsgruppen für Rheumatologie und Immunologie am Institut für Klinische Immunologie der Universität Erlangen-Nürnberg, Schwabachanlage 10, D-91054 Erlangen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 (^1)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 alpha/beta T cell receptor (TCR), CD4 or CD8, IL-2 receptor alpha-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. (^2)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.


EXPERIMENTAL PROCEDURES

Cells and Cell Culture

Peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers by density gradient centrifugation following standard procedures(15) . They were stimulated with 0.5 µg/ml purified PHA (Wellcome, Großburgwedel, Germany) and 50 units/ml human recombinant IL-2 for 5 days to obtain PHA blasts. The human T cell clones 61/39, 61/48, 68/4, and 68/5 were derived from PHA-stimulated peripheral blood mononuclear cells by immediate limiting dilution as described in (3) . Jurkat cells were derived from the original culture(16) . CEM-3.71, a CD3-positive subclone of CEM-6, HPB-ALL, Molt-3, and Molt-4 (ATCC) are uninfected T cell lines. HUT102, C91P (ATCC), and Mondi (a kind gift from Dr. Becker, Stellenbosch, SA) are transformed by HTLV-1. TAXI (a kind gift from Dr. Grassmann, Erlangen) is transformed by an H. saimiri-HTLVtax recombinant virus(17) . The simian cell lines have been described(18) . CB15 and CB23 are CD4+ human T cell lines derived from polyclonal cord blood cultures by infection with H. saimiri and have been described(1, 19) . 39-HVS and 48-HVS are permanently growing CD4+ T cell clones derived from the human T cell clones 61/39 and 61/48 by infection with H. saimiri. They have been characterized in detail(3) . 68/4-HVS and 68/5-HVS are continuously growing CD4+ T cells derived from 68/4 and 68/5 in a similar way. The H. saimiri-transformed polyclonal T cell lines CB22 and PB-M (CD4+) as well as P-1080, P-1083, P-1084, and P-1254 (CD8+) have also been tested ( (1) and (19) ; a kind gift from I. Müller-Fleckenstein, Erlangen, Germany).

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(2) 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).

Antisera and mAbs

A rabbit was immunized with a synthetic peptide corresponding to amino acids 10-29 of the ORF1 sequence (Fig. 3) coupled to ovalbumin to generate the ORF1 antiserum. Antisera to the unique regions of Src-related kinases were raised by immunizing rabbits with fusion proteins containing the unique regions of murine p56, p60, or p62 and glutathione S-transferase as described previously(2, 20, 21) . Different anti-p56 antisera and an antiserum to ERK1 were purchased from UBI (Biomol, Hamburg, Germany). Rabbit anti-mouse polyclonal antibodies were obtained from Dianova (Hamburg, Germany); the anti-phosphotyrosine mAb PY20 was purchased through ICN (Meckenheim, Germany).


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 beta-galactosidase are indicated by arrows.



Immunoprecipitations and in Vitro Kinase Assay

Cells (1.5 times 10^7/ml in serum-free medium) were lysed in TNE buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet P-40) supplemented with 1 mM sodium orthovanadate (Na(3)VO(4)), 5 mM NaF, and 10 µg/ml each of aprotinin and leupeptin (Sigma) for 20 min on ice. Lysates were cleared at 14,000 times g for 5 min, and the protein concentration in the supernatants was determined. 1 µl/sample of antiserum was added to 100-200 µg of protein for at least 1 h at 4 °C to precipitate Src-related kinases. This was followed by incubation with 50 µl of a 10% (v/v) suspension of Staphylococcus aureus particles (Pansorbin, Calbiochem). In the case of murine antibodies the S. aureus particles were preincubated with 50 µg/ml of rabbit anti-mouse antibodies and washed. The immunoprecipitates were washed five times in TNE and once in kinase buffer (20 mm MOPS, pH 7.0, and 5 mM MgCl(2)). The pellets were then incubated for 5 min in a 25-µl kinase assay mix containing 1 µM ATP (Boehringer Mannheim), 12.5 µCi [-P]ATP (Amersham Corp.) and 2 µg of rabbit muscle enolase (Sigma) in kinase buffer. The phosphotransferase reaction was stopped with sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and 5% beta-mercaptoethanol). Samples were then incubated for 30 min at room temperature and microfuged. Supernatants were boiled for 5 min before separation on 8% SDS-PAGE. Gels were dried and autoradiographed. For some experiments the in vitro kinase reaction was stopped, and the immune complexes were dissociated by boiling in 1% SDS, 10 mM Tris, and 1 mM sodium orthovanadate. The detergent was diluted at least 1:10 with 1% Triton, 10 mM Tris, 50 mM NaCl, 5 mM EDTA, and the solution was precleared from the remaining antibodies by incubation with 50 µl of S. aureus suspension followed by centrifugation. The supernatants were then incubated with the second antibody for 1 h at 4 °C and precipitated with S. aureus as described above.

In Vitro Translation of tip and Generation of Fusion Proteins

The Tip-encoding ORF1 sequences were cloned into the plasmid vector pBS (Stratagene, Heidelberg) and transcribed in vitro using bacteriophage T7 RNA polymerase (Promega/Serva, Heidelberg). The resulting RNA was translated into protein by wheat germ extracts (Promega/Serva, Heidelberg). Labeling with [S]methionine revealed a product of 40 kDa in SDS-PAGE. The protein was immunoprecipitated by the Tip antiserum but was not detected on immunoblots.

Amino-terminally truncated ORF1 (Fig. 3) and ORF2 as well as StpA coding sequences were fused to beta-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) .

Phosphopeptide Mapping and Phosphoamino Acid Analysis

For phosphopeptide mapping, proteins were cleaved using V8 protease as described in (24) . Phosphoamino acid analysis was performed according to (25) .


RESULTS

A 40-kDa Phosphoprotein Is Associated with p56 in H. saimiri-transformed T Cells

Based on the hypothesis that transformation by H. saimiri involves early signaling events in T cells we studied tyrosine phosphorylation in the transformed cell lines. In anti-p56 immune complex kinase assays we observed co-immunoprecipitation of a 40-kDa phosphoprotein (p40). This band appeared regularly in both CD4+ and CD8+ cells and not in the nontransformed parental T cell clones. In Fig. 1four different CD4+ T cell clones are compared before and after transformation. Like the nontransformed clones, uninfected resting T cells, polyclonal PHA blasts, various permanently growing human T cell lines (Jurkat, CEM-3.71, HPB-ALL, Molt-3, and Molt-4), and T cells immortalized by HTLV1 genes (HUT102, C91Pl, TAXI, and Mondi) did not show this association of the 40-kDa species with p56 (data not shown). Nonspecific precipitation of p40 by the anti-p56 antiserum was excluded by the use of other antisera to Lck (data not shown) and monoclonal antibodies directed against CD4 (Fig. 1). The 40-kDa protein is present in CD4-Lck or CD8-Lck complexes as shown by immune complex kinase assays after precipitation with antibodies against the coreceptor expressed on the cell line used ( Fig. 1and data not shown). Anti-p60 or anti-p62 antisera did not co-precipitate p40 from lysates of transformed cells (data not shown). However, expression of these enzymes in T cells is much less abundant than that of p56. Following activation of p56 by cross-linking of CD4, the phosphorylation of p40 increased similarly to that of enolase, which was added into the immune complex kinase assay as an exogenous substrate (Fig. 2) consistent with the notion that p40 is a substrate of p56.


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.



The in Vitro Translation Product of ORF1 Migrates in SDS-PAGE with an Apparent Molecular Mass of 40 kDa

Since phosphorylation of p40 was restricted to T cells infected with H. saimiri we suspected a viral protein. Only few H. saimiri genes are transcribed in transformed T cells. StpC (gene product of ORF2) was excluded, since the protein migrates at an apparent molecular mass of 20-22 kDa(26) . Another open reading frame, ORF1, is located on the same mRNA species as stpC.^2 Whereas no homologous proteins were found in the GenBank and Swiss data bases, sequence analysis of the 256 amino acid protein revealed four potential tyrosine phosphorylation sites, a region homologous to the Src family kinase regulatory domain, an SH3 binding consensus motif (XPXXPPPXP), and a hydrophobic domain at the carboxyl terminus (Fig. 3). The predicted molecular mass of the polypeptide was 28.7 kDa, but after in vitro translation the ORF1 product appeared at 40 kDa in SDS-PAGE. The discrepancy between predicted and apparent molecular mass of the ORF1 protein is probably explained by its high proline content(27) .

The p56-associated p40 Is Identical with the ORF1 Gene Product

Anti-p56 immune complexes were precipitated, and a kinase assay was performed. After disruption of the complexes by boiling in SDS, p40 could be reprecipitated by anti-phosphotyrosine antibodies, by an anti-p56 antiserum, and by a rabbit antiserum against a synthetic peptide corresponding to amino acids 10-29 of the ORF1 sequence (Fig. 4A). p40 was not seen in reprecipitations with S. aureus only or with antisera to ERK kinases or StpC (Fig. 4A). On the other hand, the antiserum to the ORF1 gene product precipitated a 40-kDa protein and associated kinase activity from H. saimiri-transformed cells (data not shown). Both p40 in p56 immunoprecipitates and p40 in anti-ORF1 immunoprecipitates were phosphorylated on tyrosine (Fig. 4B). Partial digestion with V8 protease resulted in identical phosphopeptide patterns confirming the identity of the two proteins (data not shown). Furthermore, p40 was reconstituted in lysates of nontransformed T cells by addition of the ORF1 in vitro translation product to whole cell lysates (Fig. 5). Finally, a major part of ORF1 (as indicated in Fig. 3) was expressed in bacteria as a beta-galactosidase fusion protein. Partially purified preparations of this protein were phosphorylated in kinase assays with commercially available purified Lck (UBI) (Fig. 6), anti-p56 immunoprecipitates from nontransformed T cell clones, and a recombinant GST-Lck fusion protein isolated from baculovirus-infected insect cells (data not shown). Under similar conditions the different Lck preparations did not phosphorylate beta-galactosidase fusion proteins with the ORF2-encoded proteins StpA or StpC ( Fig. 6and data not shown).


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-beta-Gal fusion protein. An Lck immunoprecipitate from uninfected T cells was added to beta-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 beta-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).


DISCUSSION

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. (^3)In addition, the phenotype of H. saimiri-immortalized T cells is remarkably stable. Cell surface markers of mature activated T cells, in particular the alpha/beta TCR-CD3 complex, CD2, CD4 or CD8, the IL-2 receptor alpha-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.


FOOTNOTES

*
The work was supported by Deutsche Forschungsgemeinschaft (Grants Bi 465/1-1 and -2 and Forschergruppe DNA-Viren des hämatopoetischen Systems), Bayerische Forschungsstiftung (Biologische Sicherheit), Bundesministerium für Forschung und Technologie (Projekt Autoimmunität and Arbeitsgruppen für Rheumatologie), and Deutscher Akademischer Austauschidieust (fellowship to B. M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
To whom correspondence should be addressed. Present address: Bernhard-Nocht-Institut für Tropenmedizin, Bernhard-Nocht-Str. 74, 20359 Hamburg, Federal Republic of Germany. Tel.: 49-40-311-82-240; Fax: 49-40-311-82-386.

(^1)
The abbreviations used are: IL-2, interleukin-2; TCR, T cell receptor; mAb, monoclonal antibody; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; LMP2A, latent membrane protein 2A; PHA, phytohemagglutinin.

(^2)
H. Fickenscher, B. Biesinger, A. Knappe, S. Wittmann, and B. Fleckenstein, submitted for publication.

(^3)
B. Simmer, B. Fleckenstein, and F. Emmrich, unpublished observations.


ACKNOWLEDGEMENTS

We thank U. Meinke for expert technical assistance, U. Friedrich for preparation of in vitro translation products, J. Vollmar for generation of the beta-Gal fusion proteins, Dr. Becker for kindly providing cell line Mondi, I. Fleckenstein for the P-1254 cell line, and Dr. R. Grassmann for the TAXI cell line. N. Wiese and K. Breuer critically reviewed the manuscript.


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