Brigitte Biesinger

The divergence between two oncogenic Herpesvirus saimiri strains in a genomic region related to the transforming phenotype

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.

Herpesvirus saimiri Tip gene causes T-cell lymphomas in transgenic mice.

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.

T-Cell Growth Transformation by Herpesvirus Saimiri Is Independent of STAT3 Activation

ABSTRACT 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.

NFκB Signaling Is Induced by the Oncoprotein Tio through Direct Interaction with TRAF6

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

Herpesvirus ateles Tio can replace herpesvirus saimiri StpC and Tip oncoproteins in growth transformation of monkey and human T cells.

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.

Activation of Noncanonical NF-κB Signaling by the Oncoprotein Tio

NF-κB 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-κB 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-κB signal requires the presence of the regulatory subunit of the inhibitor of κB kinase (IKK) complex, NF-κB essential modulator (NEMO), and the activity of its key kinase, IKKβ, 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-κB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-κB-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 IKKβ activity, yet processing to p52 remains largely unaffected by IKKβ inhibition. However, long term inhibition of IKKβ disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-κB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.

Growth Transformation of Human T Cells by Herpesvirus Saimiri Requires Multiple Tip-Lck Interaction Motifs

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.

Tyrosine Phosphorylation of the Tio Oncoprotein Is Essential for Transformation of Primary Human T Cells

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 protein StpB associates with cellular Src

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.

Noncanonical NF-κB Activation by the Oncoprotein Tio Occurs Through a Nonconserved TRAF3-Binding Motif

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.