Doris Lengenfelder

The Latency-Associated Nuclear Antigen Homolog of Herpesvirus Saimiri Inhibits Lytic Virus Replication

ABSTRACT Herpesvirus saimiri (HVS), a T-lymphotropic tumor virus of neotropical primates, and the Kaposis sarcoma-associated human herpesvirus 8 (KSHV) belong to the gamma-2-herpesvirus (Rhadinovirus) subfamily and share numerous features of genome structure and organization. The KSHV latency-associated nuclear antigen (LANA) protein appears to be relevant for viral persistence, latency, and transformation. It binds to DNA, colocalizes with viral episomal DNA, and presumably mediates efficient persistence of viral genomes. LANA further represses the transcriptional and proapoptotic activities of the p53 tumor suppressor protein. Here we report on the ORF73 gene of HVS strain C488, which is the positional and structural homolog of KSHV LANA. The ORF73 gene in OMK cells can encode a 62-kDa protein that localizes to the nucleus in a pattern similar to that of LANA. We show that the ORF73 gene product can regulate viral gene expression by acting as a transcriptional modulator of latent and lytic viral promoters. To define the HVS ORF73 function in the background of a replication-competent virus, we constructed a viral mutant that expresses ORF73 under the transcriptional control of a mifepristone (RU-486)-inducible promoter. The HVS ORF73 gene product efficiently suppresses lytic viral replication in permissive cells, indicating that it defines a critical control point between viral persistence and lytic replication.

Differential Requirement of ZAP-70 for CD2-Mediated Activation Pathways of Mature Human T Cells

This study addresses the role of the tyrosine kinase ZAP-70 in CD2-mediated T cell activation. Patients lacking ZAP-70 have few mature CD8+ T cells and high numbers of CD4+ T cells that are nonfunctional upon TCR triggering. Such a patient with a homozygous deletion in the zap-70 gene that resulted in the complete absence of ZAP-70 protein expression has been identified. Expression of the tyrosine kinases Lck, Fyn, and Syk was normal. The patient’s T cells were activated with two different pairs of mitogenic mAbs. CD2-induced phosphorylation of the ζ-chain and influx of Ca2+ was defective in the ZAP-70-deficient T cells, whereas CD2-induced phosphorylation of several other proteins, including Syk, was not affected. CD2-induced proliferation as well as production of TNF-α and IFN-γ was abrogated in ZAP-70-deficient T cells, whereas PMA plus ionomycin induced normal activation of these cells. Together, this study shows that CD2-activation triggers ZAP-70-dependent and -independent pathways. Deletion of ZAP-70 affected CD2- and CD3-mediated proliferation and cytokine production in a similar way, suggesting that one of the different CD2 pathways converges with a CD3 pathway at or upstream of the activation of ZAP-70.

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.

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.

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.

Genome-Wide Histone Acetylation Profiling of Herpesvirus saimiri in Human T Cells upon Induction with a Histone Deacetylase Inhibitor

ABSTRACT Herpesviruses establish latency in suitable host cells after primary infection and persist in their host organisms for life. Most of the viral genes are silenced during latency, also enabling the virus to escape from an immune response. This study addresses the control of viral gene silencing by epigenetic mechanisms, using Herpesvirus saimiri (HVS) as a model system. Strain C488 of this gamma-2-herpesvirus can transform human T cells to stable growth in vitro, and it persists in the nuclei of those latently infected T cells as a nonintegrating, circular, and histone-associated episome. The whole viral genome was probed for histone acetylation at high resolution by chromatin immunoprecipitation-on-chip (ChIP-on-chip) with a custom tiling microarray. Corresponding to their inactive status in human T cells, the lytic promoters consistently revealed a heterochromatic phenotype. In contrast, the left terminal region of the genome, which encodes the stably expressed oncogenes stpC and tip as well as the herpesvirus U RNAs, was associated with euchromatic histone acetylation marks representing “open” chromatin. Although HVS latency in human T lymphocytes is considered a stable and irreversible state, incubation with the histone deacetylase inhibitor trichostatin A resulted in changes reminiscent of the induction of early lytic replication. However, infectious viral particles were not produced, as the majority of cells went into apoptosis. These data show that epigenetic mechanisms are involved in both rhadinoviral latency and transition into lytic replication.

A Molecular Model for the Differential Activation of STAT3 and STAT6 by the Herpesviral Oncoprotein Tip

Constitutive STAT signaling provides growth promoting signals in many forms of malignancy. We performed molecular modeling and molecular dynamics studies of the interaction between the regulatory Src homology 2 (SH2) domains of STAT3 and 6 with phosphorylated peptides of the herpesviral oncoprotein Tip, which facilitates Src kinase mediated STAT-activation and T cell proliferation. The studies give insight into the ligand binding specificity of the STAT SH2 domains and provide the first model for the differential activation of STAT3 or STAT6 by two distinct regions of the viral Tip protein. The biological relevance of the modeled interactions was then confirmed by activation studies using corresponding recombinant oncoproteins, and finally by respective recombinant viruses. The functional data give experimental validation of the molecular dynamics study, and provide evidence for the involvement of STAT6 in the herpesvirus induced T cell proliferation.

The role of STAT6 in human T cell transformation by herpesvirus saimiri

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.

T Cell transformation by herpesvirus saimiri requires STAT5 pathways

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.