Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) drives viral B cell transformation and oncogenesis. LMP1’s transforming activity depends on its C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 recruitment to LMP1 and its role in LMP1 signalling remains elusive. Here we demonstrate that TRAF6 interacts directly with a viral TRAF6 binding motif within CTAR2. Functional and NMR studies supported by molecular modeling provide insight into the architecture of the LMP1-TRAF6 complex, which differs from that of CD40-TRAF6. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation by CTAR2 and the survival of LMP1-driven lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with the survival of EBV-transformed B cells. In this work, we identify LMP1-TRAF6 as a critical virus-host interface and validate this interaction as a potential therapeutic target in EBV-associated cancer.

Significance LINE-1 retrotransposons are mobile genetic elements that have significantly contributed to the evolution of our genome. However, unhindered LINE-1 mobility can be detrimental and cause various genetic disorders. Here, we identify the antiviral protein TRIM5α as host factor that controls LINE-1 elements and protects our genome from novel insertions. In addition, we show that TRIM5α recognizes LINE-1 ribonucleoprotein complexes as a molecular pattern and initiates innate immune signaling upon sensing. Thus, our findings confirm the role of TRIM5α as pattern recognition receptor in innate immunity and uncover its important role in safeguarding the genome against endogenous mobile genetic elements.

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.

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.

The viral protein Tio specifically stimulates noncanonical nuclear factor κB signaling through a distinct interaction with the adaptor and ubiquitin ligase TRAF3. Preventing NF-κB Pathway Crosstalk The transcription factor NF-κB (nuclear factor κB) can be activated by so-called canonical and noncanonical pathways. Activation of the noncanonical NF-κB pathway blocks the constitutive degradation of the kinase NIK (NF-κB–inducing kinase), which leads to the generation of an NF-κB subunit required for target gene expression. The viral oncoprotein Tio mimics a constitutively active receptor upstream of NF-κB signaling, and de Jong et al. found that it contains a binding motif not conserved in other proteins that bind to TRAF3 (tumor necrosis factor receptor–associated factor 3), an inhibitor of noncanonical NF-κB signaling. This TRAF3-binding motif enabled Tio to specifically activate noncanonical NF-κB signaling without triggering crosstalk with the canonical pathway. Tio signaling did not result in TRAF3 degradation; rather, it induced the sequestration of a TRAF3-containing degradative complex from NIK to stimulate the noncanonical pathway. These data suggest that Tio might be used as a tool to examine the specific activation of genes targeted by noncanonical NF-κB signaling in the context of viral transformation. 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.

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.

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.

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.

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.

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.

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-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.

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-kappa B 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-kappa B activation, dominant negative I kappa B alpha and constitutively active IKK2, established the importance of NF-kappa B in StpC-mediated upregulation of IFN-gamma production. These observations suggest that NF-kappa B induction by StpC contributes to the Th1-like phenotype of virus-transformed human T cells.

The transcription factor NF kappa B is a major regulator of genes involved in inflammation and oncogenesis. NF kappa B 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 I kappa B, the administrator of NF kappa B activity. Here we report on a strong NF kappa B activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NF kappa B 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 NF kappa B activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NF kappa B 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 NF kappa B through an oncoprotein.

ABSTRACT 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.

ABSTRACT 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.

ABSTRACT 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.

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.