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The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) drives viral B cell transformation and oncogenesis. LMP1's transforming activity depends on its cytoplasmic C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 interaction with LMP1 and its critical role for LMP1 signaling has remained elusive. Here we demonstrate that TRAF6 interacts directly with a novel viral TRAF6 binding motif within CTAR2. Structural modeling supported by NMR and functional studies provides insight into the molecular architecture of the LMP1-TRAF6 complex and reveals substantial differences to CD40-TRAF6 interaction. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation and survival of LMP1-driven B cell lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with proliferation of EBV-transformed B cells. We identify LMP1-TRAF6 as critical virus-host interface and validate this interaction as novel therapeutic target against EBV.

Mobile genetic elements have significantly shaped our genomic landscape. LINE-1 retroelements are the only autonomously active elements left in the human genome. Since new insertions can have detrimental consequences, cells need to efficiently control LINE-1 retrotransposition. Here, we demonstrate that the intrinsic immune factor TRIM5α senses and restricts LINE-1 retroelements. Previously, rhesus TRIM5α has been shown to efficiently block HIV-1 replication, while human TRIM5α was found to be less active. Surprisingly, we found that both human and rhesus TRIM5α efficiently repress human LINE-1 retrotransposition. TRIM5α interacts with LINE-1 ribonucleoprotein complexes in the cytoplasm, which is essential for restriction. In line with its postulated role as pattern recognition receptor, we show that TRIM5α also induces innate immune signaling upon interaction with LINE-1 ribonucleoprotein complexes. The signaling events activate the transcription factors AP-1 and NF-κB, leading to the down-regulation of LINE-1 promoter activity. Together, our findings identify LINE-1 as important target of human TRIM5α, which restricts and senses LINE-1 via two distinct mechanisms. Our results corroborate TRIM5α as pattern recognition receptor and shed light on its previously undescribed activity against mobile genetic elements, such as LINE-1, to protect the integrity of our genome.

CD83 is one of the best known surface markers for mature human dendritic cells (DCs). The full-length 45 kDa type-I membrane-bound form (mbCD83) is strongly glycosylated upon DCs maturation. As co-stimulatory properties of CD83 are attributed to mbCD83 surface expression is required for efficient T-cell stimulation by mature DCs. By yeast two-hybrid screening, we were able to identify GRASP55 as interaction partner of CD83. DCs maturation induces endogenous CD83 protein expression with simultaneous regulation of CD83 glycosylation, interaction and co-localization with GRASP55 and CD83 surface exposure. GRASP55 is especially known for its role in maintaining Golgi architecture, but also plays a role in Golgi transport of specific cargo proteins bearing a C-terminal valine residue. Here we additionally demonstrate that binding of CD83 and GRASP55 rely on the C-terminal TELV-motif of CD83. Mutation of this TELV-motif not only disrupted binding to GRASP55, but also altered the glycosylation pattern of CD83 and reduced its membrane expression. Here we show for the first time that GRASP55 interacts with CD83 shortly after induction of DC maturation and that this interaction plays a role in CD83 glycosylation as well as in surface expression of CD83 on DCs.

NF-kappaB transcription factors are key regulators of cellular proliferation and frequently contribute to oncogenesis. The herpesviral oncoprotein Tio, which promotes growth transformation of human T cells in a recombinant herpesvirus saimiri background, potently induces canonical NF-kappaB signaling through membrane recruitment of the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, we show that, in addition to Tio-TRAF6 interaction, the Tio-induced canonical NF-kappaB signal requires the presence of the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO), and the activity of its key kinase, IKKbeta, to up-regulate expression of endogenous cellular inhibitor of apoptosis 2 (cIAP2) and interleukin 8 (IL-8) proteins. Dependent on TRAF6 and NEMO, Tio enhances the expression of the noncanonical NF-kappaB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-kappaB-inducing kinase (NIK). Concomitantly, Tio induces efficient processing of the p100 precursor molecule to its active form, p52, as well as DNA binding of nuclear p52 and RelB. In human T cells transformed by infection with a Tio-recombinant virus, sustained expression of p100, RelB, and cIAP2 depends on IKKbeta activity, yet processing to p52 remains largely unaffected by IKKbeta inhibition. However, long term inhibition of IKKbeta disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-kappaB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.

Interferon-γ (IFN-γ) is an essential regulator of innate and adaptive immune responses and a hallmark of the Th1 T-cell subset. It is produced at high levels by human T lymphocytes upon transformation with Herpesvirus saimiri, which depends on the expression of the viral oncoproteins saimiri transformation-associated protein of subgroup C (StpC) and tyrosine kinase-interacting protein (Tip). Here, we show that IFN-γ production was induced by Tip in Jurkat T cells. StpC by itself did not affect IFN-γ expression, but enhanced the effect of Tip. Our results substantiated the findings that StpC induces NF-κB activation and demonstrated that other transcription factors, including NFAT, AP-1 and serum response element regulators, were not activated by StpC in unstimulated T cells. Studies using StpC mutants deficient in NF-κB activation, dominant negative IκBα and constitutively active IKK2, established the importance of NF-κB in StpC-mediated upregulation of IFN-γ production. These observations suggest that NF-κB induction by StpC contributes to the Th1-like phenotype of virus-transformed human T cells.

Adult T-cell leukemia/lymphoma is a highly infiltrative neoplasia of CD4+ T-lymphocytes that occurs in about 5% of carriers infected with the deltaretrovirus human T-cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax perturbs cellular signaling pathways leading to upregulation of host cell factors, amongst them the actin-bundling protein Fascin, an invasion marker of several types of cancer. However, transcriptional regulation of Fascin by Tax is poorly understood. In this study, we identified a triple mode of transcriptional induction of Fascin by Tax, which requires (1) NF-κB-dependent promoter activation, (2) a Tax-responsive region in the Fascin promoter, and (3) a promoter-independent mechanism sensitive to the Src family kinase inhibitor PP2. Thus, Tax regulates Fascin by a multitude of signals. Beyond, using Tax-expressing and virus-transformed lymphocytes as a model system, our study is the first to identify the invasion marker Fascin as a novel target of PP2, an inhibitor of metastasis.

The transcription factor NFkappaB is a major regulator of genes involved in inflammation and oncogenesis. NFkappaB is induced upon stimulation of cellular receptors coupled to different intracellular signaling molecules. Further downstream, TRAF6 links at least two receptor pathways to take control of IkappaB, the administrator of NFkappaB activity. Here we report on a strong NFkappaB activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NFkappaB induction by Tio is independent of Src-kinase interaction and tyrosine phosphorylation of Tio. Mutation of a glutamic acid-rich motif at the N terminus of Tio, corresponding to a TRAF6 consensus binding motif, completely abrogated NFkappaB activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NFkappaB activation. Furthermore, we provide evidence that TRAF6 directly binds to the Tio oncoprotein. Identification of TRAF6 as the direct target of Tio describes a novel mechanism for the constitutive activation of NFkappaB through an oncoprotein.

Protein tyrosine kinases (PTKs) are critically involved in signaling pathways that regulate cell growth, differentiation, activation, and transformation. It is not surprising, therefore, that viruses acquire effector molecules targeting these kinases to ensure their own replication and/or persistence. This review summarizes our current knowledge on Lck, a member of the Src family of PTK, and its viral interaction partners. Lck plays a key role in T lymphocyte activation and differentiation. It is associated with a variety of cell surface receptors and is critical for signal transduction from the T-cell antigen receptor (TCR). Consequently, Lck is targeted by regulatory proteins of T-lymphotropic viruses, especially by the Herpesvirus saimiri (HVS) tyrosine kinase interacting protein (Tip). This oncoprotein physically interacts with Lck in HVS transformed T cells and has an impact on its catalytic activity. However, while Tip inhibits Lck activity in stably expressing cell lines, opposite effects were observed in several in vitro systems. At least in part, this complex situation may be related to the bipartite nature of the interaction surface of the two proteins. Studies on the interrelationships between Lck and its viral partners contribute to the understanding of the mechanisms of T-cell growth regulation, in general, and of viral pathogenicity in particular. In addition, understanding the regulation of Lck activity by viral proteins may serve as a basis for the development of new drugs capable of modifying Lck activity in different pathological situations.

Herpesvirus saimiri (HVS) causes T-lymphoproliferative dis-$borders in several New World and Old World primate species and in certain rabbits.In vitro infection leads to permanent growth of primary T cells of primate and human origins. The transformation-relevant proteins of HVS inter-$bact with cellular proto-oncoproteins which results in cell growth transformation. In addition, virus-encoded cellular homologues may contribute to transformation or persistence of HVS by altering cellular signal transduction and deregulating cell growth control. Because of the presence of a permissive cell culture system and in vitro Land in vivo transformation assays, HVS provides a unique opportunity to investigate the mechanisms of cancer induction by oncogenic herpesviruses.

Herpesvirus ateles is a gamma-2-herpesvirus which naturally infects spider monkeys ( Ateles spp.) and causes malignant lymphoproliferative disorders in various other New World primates. The genomic sequence of herpesvirus ateles strain 73 revealed a close relationship to herpesvirus saimiri, with a high degree of variability within the left terminus of the coding region. A spliced mRNA transcribed from this region was detected in New World monkey T-cell lines transformed by herpesvirus ateles in vitro or derived from T cells of infected Saguinus oedipus . The encoded viral protein, termed Tio, shows restricted homology to the oncoprotein StpC and to the tyrosine kinase-interacting protein Tip, two gene products responsible for the T-cell-transforming and oncogenic phenotype of herpesvirus saimiri group C strains. Tio was detectable in lysates of the transformed T lymphocytes. Dimer formation was observed after expression of recombinant Tio. After cotransfection, Tio was phosphorylated in vivo by the protein tyrosine kinases Lck and Src and less efficiently by Fyn. Stable complexes of these Src family kinases with the viral protein were detected in lysates of the transfected cells. Binding analyses indicated a direct interaction of Tio with the SH3 domains of Lyn, Hck, Lck, Src, Fyn, and Yes. In addition, tyrosine-phosphorylated Tio bound to the SH2 domains of Lck, Src, or Fyn. Thus, herpesvirus ateles-encoded Tio may contribute to viral T-cell transformation by influencing the function of Src family kinases.

Members of the nuclear factor κB (NF-κB) family of transcription factors regulate many cellular functions. Activation of NF-κB signaling is commonly classified as occurring through canonical or noncanonical pathways. Most NF-κB-inducing stimuli, including the viral oncoprotein Tio, lead to a concerted activation of both NF-κB pathways; however, extensive crosstalk at multiple levels between these signaling cascades restricts the ability to discriminate between the canonical and the noncanonical effects. We showed that noncanonical NF-κB activation by Tio depends on a distinct sequence motif that directly recruits tumor necrosis factor receptor-associated factor 3 (TRAF3). Through its TRAF3-binding motif, Tio triggered a ubiquitin-independent depletion of TRAF3 from the cytosol, which prevented TRAF3 from inhibiting signaling through the noncanonical NF-κB cascade. Furthermore, the Tio-TRAF3 interaction did not affect components of the canonical NF-κB signaling pathway or the expression of target genes; thus, Tio induced noncanonical NF-κB independently of crosstalk with the canonical pathway. Together, these data identify a distinct molecular mechanism of noncanonical NF-κB activation that should enable studies into the particular functions of this pathway.

A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.

Serum response factor (SRF) acts as a multifunctional transcription factor regulated by mutually exclusive interactions with ternary complex factors (TCFs) or myocardin-related transcription factors (MRTFs). Binding of Rho- and actin-regulated MRTF:SRF complexes to target gene promoters requires an SRF-binding site only, whereas MAPK-regulated TCF:SRF complexes in addition rely on flanking sequences present in the serum response element (SRE). Here, we report on the activation of an SRE luciferase reporter by Tip, the viral oncoprotein essentially contributing to human T-cell transformation by Herpesvirus saimiri. SRE activation in Tip-expressing Jurkat T cells could not be attributed to triggering of the MAPK pathway. Therefore, we further analyzed the contribution of MRTF complexes. Indeed, Tip also activated a reporter construct responsive to MRTF:SRF. Activation of this reporter was abrogated by overexpression of a dominant negative mutant of the MRTF-family member MAL. Moreover, enrichment of monomeric actin suppressed the Tip-induced reporter activity. Further upstream, the Rho-family GTPase Rac, was found to be required for MRTF:SRF reporter activation by Tip. Initiation of this pathway was strictly dependent on Tip's ability to interact with Lck and on the activity of this Src-family kinase. Independent of Tip, T-cell stimulation orchestrates Src-family kinase, MAPK and actin pathways to induce SRF. These findings establish actin-regulated transcription in human T cells and suggest its role in viral oncogenesis.

The potential of Herpesvirus saimiri (HVS) subgroups A, B and C and Herpesvirus ateles (HVA) to transform primary T cells to permanent growth in vitro is restricted by the primate host species and by viral variability represented by distinct viral oncoproteins. We now addressed the relation between the transforming potential of the different viruses and the signaling pathways activated by transiently expressed oncoproteins. Marmoset lymphocytes were transformed by all HVS subgroups as well as HVA, while transformation of human cells was restricted to HVS-C and, unexpectedly, HVA. NF-κB and Src-family kinase (SFK) activity was required for survival of all transformed lymphocytes. Accordingly, NF-κB was induced by oncoproteins of all viruses. In contrast, SFK-related signaling was detectable only for oncoproteins of HVS-C and HVA. Thus, the restricted transformation of human lymphocytes likely correlates with the specific SFK targeting by these oncoproteins. These results will enable further studies into novel SFK effector mechanisms relevant for T-cell proliferation.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.

Induction of T-cell lymphomas and T-cell growth transformation by Herpesvirus saimiri strain C-488 depend on the saimiri transformation-associated protein of subgroup C (StpC). Previous studies identified the transcription factor NF-κB as the major cellular target of StpC. NF-κB activation relies on a TRAF binding motif in StpC and was enhanced by coexpression of constitutively active Ras or Raf. Concomitantly, StpC repressed Ras-mediated ERK and AP-1 activation. Nevertheless, we now found that specific inhibitors of MEK as well as ERK abrogated cooperative NF-κB activation. Triggering the ERK pathway by external stimuli, e.g. PMA, also enhanced StpC-induced NF-κB activity, however, with a significant delay relative to ERK1/2 phosphorylation. These observations suggested that ERK activity regulates the expression of proteins limiting StpC's capacity to induce NF-κB. Westernblot analyses of proteins representing the classical and alternative pathways of NF-κB activation revealed that StpC cooperates with Ras and even more with PMA to upregulate the expression and nuclear localization of RelB and NF-κB2/p52; furthermore, StpC coimmunoprecipitated TRAF2, but not TRAF6, Ras or Raf. In summary, these data suggest that ERK-inducing signaling pathways support NF-κB activation by StpC through an enhanced expression of NF-κB proteins utilized by the alternative pathway, which is triggered by StpC:TRAF2 complexes. Future studies will have to address the relevance of the enhancing effect for the proliferation of Herpesvirus saimiri-transformed human T lymphocytes.

Aberrant growth stimulation by Signal Transducers and Activators of Transcription (STAT) is implicated in human carcinogenesis. The viral effector oncoprotein Tip, which binds to and is phosphorylated by the T-cell lymphocyte kinase Lck, is crucial in the activation of the cellular STAT5 signalling pathway in Herpesvirus-transformed T-cell lines, a model for malignant T-cell proliferation. While Tip-Lck interaction is required for transformation by HVS, mutation of Tip tyrosine residues had distinct effects. Two defined mutations of Tip can direct the phosphorylation and activation of either STAT3 or STAT5 by Lck. Tip Y114 mutation to phenylalanine (TipY114F) abolished the constitutive STAT3 activation observed in HVS-wildtype transformed T cells. Conversely, TipY114F enhanced the efficiency of human T cell transformation in absence of exogenous interleukin-2 (IL-2). In contrast, mutation of the major phosphorylation site, Y127, in Tip is compatible with viral transformation only when IL-2 is supplemented. This growth factor requirement correlated with STAT5 activation by Lck and Tip. Our current work focuses on the synergy among Tip, Lck, STAT5 and JAK family kinases. Interaction analysis will address whether Tip or the Y127F mutant mediate interactions between STAT5 and Lck or other Src family members. The role of the JAK-STAT pathway was elucidated using specific JAK inhibitors. We assume that, for viral transformation, Tip preferentially targets and reprograms the STAT5 pathway, which is central for T-cell growth and homeostasis. Comparable modulation of the STAT5 signalling may be involved in other forms of malignancy and cell differentiation; their analysis may therefore be a useful model for the development of new therapeutics.

Interferon-gamma (IFN-gamma) is an essential regulator of innate and adaptive immune responses and a hallmark of the Th1 T-cell subset. It is produced at high levels by human T lymphocytes upon transformation with Herpesvirus saimiri, which depends on the expression of the viral oncoproteins saimiri transformation-associated protein of subgroup C (StpC) and tyrosine kinase-interacting protein (Tip). Here, we show that IFN-gamma production was induced by Tip in Jurkat T cells. StpC by itself did not affect IFN-gamma expression, but enhanced the effect of Tip. Our results substantiated the findings that StpC induces NF-kappaB activation and demonstrated that other transcription factors, including NFAT, AP-1 and serum response element regulators, were not activated by StpC in unstimulated T cells. Studies using StpC mutants deficient in NF-kappaB activation, dominant negative IkappaBalpha and constitutively active IKK2, established the importance of NF-kappaB in StpC-mediated upregulation of IFN-gamma production. These observations suggest that NF-kappaB induction by StpC contributes to the Th1-like phenotype of virus-transformed human T cells.

Lymphoma induction and T-cell transformation by herpesvirus saimiri strain C488 depends on two viral oncoproteins, StpC and Tip. The major interaction partner of Tip is the protein tyrosine kinase Lck, a key regulator of T-cell activation. The Lck binding domain (LBD) of Tip comprises two interaction motifs, a proline-rich SH3 domain-binding sequence (SH3B) and a region with homology to the C terminus of Src family kinase domains (CSKH). In addition, biophysical binding analyses with purified Lck-SH2 domain suggest the phosphorylated tyrosine residue 127 of Tip (pY127) as a potential third Lck interaction site. Here, we addressed the relevance of the individual binding motifs, SH3B, CSKH, and pY127, for Tip-Lck interaction and for human T-cell transformation. Both motifs within the LBD displayed Lck binding activities and cooperated to achieve a highly efficient interaction, while pY127, the major tyrosine phosphorylation site of Tip, did not enhance Lck binding in T cells. Herpesvirus saimiri strain C488 recombinants lacking one or both LBD motifs of Tip lost their transforming potential on human cord blood lymphocytes. Recombinant virus expressing Tip with a mutation at position Y127 was still able to transform human T lymphocytes but, in contrast to wild-type virus, was strictly dependent on exogenous interleukin-2. Thus, the strong Lck binding mediated by cooperation of both LBD motifs was essential for the transformation of human T cells by herpesvirus saimiri C488. The major tyrosine phosphorylation site Y127 of Tip was particularly required for transformation in the absence of exogenous interleukin-2, suggesting its involvement in cytokine signaling pathways.

Human T cells are transformed to antigen-independent permanent growth in vitro upon infection with herpesvirus saimiri subgroup C strains. The viral oncoproteins required for this process, StpC and Tip, could be replaced by Tio, the oncoprotein of herpesvirus ateles. Here we demonstrate that proliferation of lymphocytes transformed with Tio-recombinant herpesvirus saimiri required the activity of Src family kinases. Src kinases had previously been identified as interaction partners of Tio. This interaction was now shown to be independent of any of the four tyrosine residues of Tio but to be dependent on an SH3-binding motif. Mutations within this motif abrogated the transforming capabilities of Tio-recombinant herpesvirus saimiri. Furthermore, kinase interaction resulted in the phosphorylation of Tio on a single tyrosine residue at position 136. Mutation of this residue in the viral context revealed that this phosphorylation site, but none of the other tyrosine residues, was required for T-cell transformation. These data indicate that the interaction of Tio with a Src kinase is essential for both the initiation and the maintenance of T-cell transformation by recombinant herpesvirus saimiri. The requirement for the tyrosine phosphorylation site at position 136 suggests a role for Tio beyond simple deregulation of the kinase.

Herpesvirus saimiri (saimirine herpesvirus 2) (HVS), a T-lymphotropic tumor virus, induces lymphoproliferative disease in several species of New World primates. In addition, strains of HVS subgroup C are able to transform T cells of Old World primates, including humans, to permanently growing T-cell lines. In concert with the Stp oncoprotein, the tyrosine kinase-interacting protein (Tip) of HVS C488 is required for T-cell transformation in vitro and lymphoma induction in vivo. Tip was previously shown to interact with the protein tyrosine kinase Lck. Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis and has also been detected in HVS-transformed T-cell lines. Furthermore, Tip contains a putative consensus YXPQ binding motif for the SH2 ( src homology 2) domains of STAT1 and STAT3. Tip tyrosine phosphorylation at this site was required for binding of STATs and induction of STAT-dependent transcription. Here we sought to address the relevance of STAT activation for transformation of human T cells by introducing a tyrosine-to-phenylalanine mutation in the YXPQ motif of Tip of HVS C488. Unexpectedly, the recombinant virus was still able to transform human T lymphocytes, but it had lost its capability to activate STAT3 as well as STAT1. This demonstrates that growth transformation by HVS is independent of STAT3 activation.

Herpesvirus saimiri group C strains are capable of transforming human and simian T-lymphocyte populations to permanent antigen-independent growth. Two viral oncoproteins, StpC and Tip, that are encoded by a single bicistronic mRNA, act in concert to mediate this phenotype. A closely related New World monkey herpesvirus, herpesvirus ateles, transcribes a single spliced mRNA at an equivalent genome locus. The encoded protein, Tio, has sequence homologies to both StpC and Tip. We inserted the tio sequence of herpesvirus ateles strain 73 into a recombinant herpesvirus saimiri C488 lacking its own stpC / tip oncogene. Simian as well as human T lymphocytes were growth transformed by the chimeric Tio-expressing viruses. Thus, a single herpesvirus protein appears to be responsible for the oncogenic effects of herpesvirus ateles.

Herpesvirus saimiri codes for a tyrosine kinase interacting protein (Tip) that interacts with both the SH3 domain and the kinase domain of the T-cell-specific tyrosine kinase Lck via two separate motifs. The activation of Lck by Tip is considered as a key event in the transformation of human T-lymphocytes during herpesviral infection. We investigated the interaction of proline-rich Tip peptides with the LckSH3 domain starting with the structural characterization of the unbound interaction partners. The solution structure of the LckSH3 was determined by heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy using 44 residual dipolar couplings in addition to the conventional experimental restraints. Circular dichroism spectroscopy proved that the polyproline helix of Tip is already formed prior to SH3 binding and is conformationally stable. NMR titration experiments point out three major regions of the Tip-Lck interaction comprising the RT loop, the n-src loop, and a helical turn preceding the last strand of the beta-sheet. Further changes of the chemical shifts were observed for the N- and C-terminal beta-strands of the SH3 domain, indicating additional contacts outside the proline-rich segment or subtle structural rearrangements transmitted from the binding site of the proline helix. Fluorescence spectroscopy shows that Tip binds to the SH3 domains of several Src kinases (Lck, Hck, Lyn, Src, Fyn, Yes), exhibiting the highest affinities for Lyn, Hck, and Lck.

Efficiency of lymphoma induction by herpesvirus saimiri (HVS) isolates correlates with the genetically defined viral subgroups A, B, and C. To compare subgroup-specific effects, highly susceptible tamarins were infected with HVS strain A-11, B-SMHI, or C-488. All animals developed T-cell lymphomas indistinguishable with respect to clinical, pathological, and virological parameters. Ex vivo T-cell lines were established readily from the HVS C-488 animal, less efficiently in the presence of HVS A-11, and from only a single HVS B-SMHI sample. These cultivated cells revealed strain-specific biochemical characteristics. HVS A-11 strongly induced the expression of tyrosine kinase Lyn. HVS C-488 led to the activation of STAT3, which is most likely linked to the association of virus-encoded Tip with tyrosine kinase Lck. The lack of these activities in HVS B-SMHI-transformed cells may correlate with the reduced oncogenic phenotype of this virus in species other than tamarins.

New World primates develop T-cell lymphomas on infection with Herpesvirus saimiri. To investigate the oncogenic potential of the Tip gene of Herpesvirus saimiri strain C488, we tried to establish transgenic mice that should express Tip under control of a constitutive promoter. Although transgene-positive embryos were found, lines could not be established. However, using a system in which the transgene has to be activated by a Cre recombinase-mediated deletion, we were able to obtain several Tip transgenic lines. At high expression levels, the mice developed T-cell lymphomas. Thus, Tip can induce lymphomas and is therefore very likely responsible for the oncogenicity of Herpesvirus saimiri.

Subgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.

Herpesvirus saimiri strain C488, a T-cell tumor virus of New World primates, transforms human T lymphocytes to stable interleukin-2-dependent growth without need for further stimulation by antigen or mitogen. The transformed cell lines show the phenotype of activated mature T cells and retain many essential features of the primary parental cells, e.g., antigen specificity. In contrast to transformed New World monkey T cells, the human lines do not support lytic growth of the virus, even after chemical stimulation. Here we show that many viral genes remain silent during episomal persistence. However, the viral oncogene stpC is predominantly transcribed and translated to a stable cytoplasmic protein of 20 kDa that is heterogeneously expressed in individual cells. This 1.7-kb mRNA is bicistronic, encoding also Tip, a viral protein interacting with the T-cell-specific tyrosine kinase Lck. stpC/tip transcripts are heavily induced upon stimulation by mitogen or phorbol ester. Block of protein synthesis does not abolish transcription: treatment with cycloheximide greatly induces stpC/tip mRNA levels. Thus, this gene complex is regulated similarly to early T-cell activation genes. Constitutive and induced expression engage different transcription start sites. The T-cell regulation of the viral genes stpC and tip may contribute to the T-cell tropism of growth transformation by herpesvirus saimiri.

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 p56lck. 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.

Human peripheral blood T lymphocytes are readily transformed to continuous growth by Herpesvirus saimiri subgroup C strains. The immortalized cells express the phenotype of mature activated T cells and bear either CD4 or CD8 surface markers. Here we report that Herpesvirus saimiri transformed CD4 + cell lines are highly susceptible to infection with human immunodeficiency viruses types 1 and 2. The prototype viruses HIV-1 III8 and HIV-2 ROD replicated rapidly and caused cell death within 14 days. These cell lines did also support growth of a poorly replicating HIV-2 strain (HIV-2 NEP ) and of primary clinical isolates. Thus H. saimiri transformed T cells represent a new system for HIV propagation and isolation, especially for HIV-2 strains with restricted cell tropism. These cells should be considered as an alternative approach in cases where conventional attempts fail.

Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies.

Herpesvirus saimiri (HVS) induces T-cell lymphoma in New World primates. HVS subgroup C strains growth transform human T-cells in vitro. Oncoproteins StpC and Tip are essential for transformation. The lymphocyte tyrosine kinase Lck is the major interaction partner of Tip and phosphorylates Tip at specific tyrosine residues. We find that STAT6 is activated by Tip together with Lck, requiring either Tip residues, Y114 or Y127 for increased activation and both for full activation. Our analysis addresses whether Tip or the Y114F and/or Y127F mutant mediate interactions between STAT6 and Lck or other Src family members. The components are expressed and purified using the Strep-tag system to identify additional factors involved in complex formation by affinity chromatography. Furthermore, respective fluorescent fusion proteins are made to observe colocalization patterns and interaction within living T cells by fluorescence resonance energy transfer. These studies on the association of Tip, Lck, and STATs can resolve further regulatory mechanisms involved in viral transformation process.

Herpesvirus saimiri is an oncogenic herpesvirus that induces rapidly progressing lymphomas in New World primates. Using retrovirus vectors for gene transfer, specific open reading frames of H. saimiri were tested for their ability to transform rodent cells in culture. One open reading frame, designated STP-C488 (for saimiri-transformation-associated protein of the subgroup C strain 488), phenotypically transformed Rat-1 cells, resulting in formation of foci, growth at reduced serum concentration, and growth to higher cell densities. Cells transformed by STP-C488 formed invasive tumors in nude mice. The STP-A11 reading frame of strain 11 (subgroup A) was much less potent in its transforming ability than STP-C488. These results demonstrate the oncogene nature of these two open reading frames and provide a means for studying their transforming functions independent of the rest of the H. saimiri genome.

Herpesvirus saimiri strains can be divided into at least three subgroups (A, B, C) based on sequence divergence at the left end of viral unique sequence DNA. Strains of subgroups A and C are highly oncogenic and readily transform simian T-lymphocytes in vitro to interleukin-2 independent growth, while subgroup B strains do not. A left terminal reading frame of a H. saimiri subgroup A strain was shown previously to correlate with the oncogenic phenotype and in vitro transforming potential; the deduced polypeptide was termed STP-A. Furthermore, this same region contains an open reading frame (ORF) for dihydrofolate reductase (DHFR) and genes for five virus-specific U RNAs (HSURs). We now show by sequence analysis of the corresponding region in a subgroup C strain that DHFR and HSUR genes are present in both virus subgroups; however, no sequence homologous to the STP-A reading frame was found in this subgroup C virus. At a position and orientation similar to STP-A, two ORFs were found for peptides sharing a putative transmembrane domain. One of them encodes a peptide with collagen-like repetitions. In addition to the lack of similarity to STP-A, these two reading frames also did not show any similarity to known oncogenes. The organization of sequences at the left junction of unique L- and repetitive H-DNA of H. saimiri suggests frequent recombinational events, possibly accelerating the uptake of foreign genes by the virus.

This study addresses the timing of gammaherpesviral episomal DNA replication with respect to the cell cycle. For the first time we analyzed a rhadinovirus, the prototype Herpesvirus saimiri (HVS), and compared it to the lymphocryptovirus Epstein-Barr virus (EBV). Newly synthesized DNA of latently infected B- or T-cells was first BrdU-labeled; then we sorted the cells corresponding to cell cycle phases G(0/1), G(2/M), and S (4 fractions S(1)-S(4)) and performed anti-BrdU chromatin immunoprecipitation. Next, DNA of different viral gene loci was quantitatively detected together with cellular control genes of known replication time. The sensitive technique is further enhanced by an internal coprecipitation standard for increased precision. Both gammaherpesviruses replicated very early in S-phase, together with cellular euchromatin. Our work suggests that early S-phase DNA replication is a general characteristic of episomal herpesviral genomes.

Herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) are T-lymphotropic viruses common in certain New World primates. Both viruses appear not to be pathogenic in their natural host species, squirrel monkeys (Saimiri sciureus) and spider monkeys (Ateles spp.), respectively. However, they can induce rapidly progressing T-cell lymphomas in a wide spectrum of South American monkeys (reviewed in Flecken-stein and Desrosiers 1982), and both viruses are capable of transforming T cells of marmoset monkeys in vitro (Falk et al. 1978; Schirm et al. 1984; Desrosiers et al. 1986). The herpesvirus group is in general highly heterogeneous with respect to the molecular structure of genomic DNA and pathogenic properties.

Based on sequence divergence in the transformation-relevant region, herpesvirus saimiri strains are classified into three subgroups. Only members of subgroup C transform human T lymphocytes to continuous interleukin-2-dependent growth in culture. In this study, human cord blood T cells were immortalized by using different subgroup C strains (C488, C484, and C139). The resulting T-cell lines represented different types of T-cell clones. They were either CD4+ or CD8+ and expressed either the alphabeta or the gammadelta type of T-cell receptors. If transformed by the same virus strain, alphabeta and gammadelta clones were similar with respect to viral persistence, virus gene expression, proliferation, and Th1-type cytokine production. However, major differences were observed in T cells immortalized by different subgroup C strains. Strain C139 persisted at low copy number, compared to the high copy number of prototype C488. The transformation-associated genes stpC and tip of strain C488 were strongly induced after T-cell stimulation. The homologous genes of strain C139 were only weakly expressed and not induced after activation. After CD2 ligation, the C488-transformed T cells produced interleukin-2, whereas the C139-transformed cells did not. Correspondingly, the C139-transformed T cells were less sensitive to cyclosporin A. Sequence comparison from different subgroup C strains revealed a variability of the stpC/tip promoter region and of the Lck-binding viral protein Tip. Thus, closely related subgroup C strains of herpesvirus saimiri cause major differences in the functional phenotype of growth-transformed human T cells.