Jens-Christian Albrecht

Herpesvirus saimiri has a gene specifying a homologue of the cellular membrane glycoprotein CD59

Herpesvirus saimiri (HSV) is a T-lymphotropic tumor virus that causes fulminant lymphomas and leukemias in various New World primates other than its natural host, the squirrel monkey (Saimiri sciureus). In the course of completing the nucleotide sequence of its genome, we identified an open reading frame of 363 nucleotides, designated HVS-15, that has no detectable homology to any other viral sequences to date. HVS-15 encodes a 121-amino-acid protein which shows significant similarities to human CD59, a phosphatidyl-inositol-glycan-anchored glycoprotein involved in T-cell activation and restriction of complement-mediated lysis. The predicted HVS-15 gene product is more similar to human CD59 than to the related murine Ly-6 antigens. A nucleotide sequence identity of 64% was found between HVS-15 and the CD59 reading frame, and a 48% identity exists between the corresponding protein sequences. The comparison of the amino acid sequences revealed a number of conserved structural features such as a similar pattern of hydrophobic termini and an identical cysteine skeleton.

Primary Structure of the Herpesvirus Ateles Genome

ABSTRACT Herpesvirus ateles is an agent indigenous to spider monkeys (Ateles spp.) and causes fulminant lymphomas in various New World primates. Structural and genetic relatedness led to the classification of this virus as a member of the genusRhadinovirus. It is most closely related toHerpesvirus saimiri. The 108,409-bp light DNA segment of the herpesvirus ateles strain 73 genome has two genes for U-RNA-like transcripts and 73 open reading frames, of which at least 6 show significant homologies to cellular genes (encoding complement control proteins, apoptosis-regulatory proteins, D-type cyclins, interleukin-8 receptors, and enzymes involved in nucleotide metabolism). The left terminal region of the light DNA segment bears the putative rhadinovirus oncogene tio.

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.

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.

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.

Actin-dependent activation of serum response factor in T cells by the viral oncoprotein tip

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 Tips 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.

NF-κB activation by the viral oncoprotein StpC enhances IFN-γ production in T cells

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.

Species restriction of Herpesvirus saimiri and Herpesvirus ateles: Human lymphocyte transformation correlates with distinct signaling properties of viral oncoproteins

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.