Bernhard Fleckenstein

Cell Surface Heparan Sulfate Is a Receptor for Human Herpesvirus 8 and Interacts with Envelope Glycoprotein K8.1

ABSTRACT An immunodominant envelope glycoprotein is encoded by the human herpesvirus 8 (HHV-8) (also termed Kaposis sarcoma-associated herpesvirus) K8.1 gene. The functional role of glycoprotein K8.1 is unknown, and recognizable sequence homology to K8.1 is not detectable in the genomes of most other closely related gammaherpesviruses, such as herpesvirus saimiri or Epstein-Barr virus. In search for a possible function for K8.1, we expressed the ectodomain of K8.1 fused to the Fc part of human immunoglobulin G1 (K8.1ΔTMFc). K8.1ΔTMFc specifically bound to the surface of cells expressing glycosaminoglycans but not to mutant cell lines negative for the expression of heparan sulfate proteoglycans. Binding of K8.1ΔTMFc to mammalian cells could be blocked by heparin. Interestingly, the infection of primary human endothelial cells by HHV-8 could also be blocked by similar concentrations of heparin. The specificity and affinity of these interactions were then determined by surface plasmon resonance measurements using immobilized heparin and soluble K8.1. This revealed that K8.1 binds to heparin with an affinity comparable to that of glycoproteins B and C of herpes simplex virus, which are known to be involved in target cell recognition by binding to cell surface proteoglycans, especially heparan sulfate. We conclude that cell surface glycosaminoglycans play a crucial role in HHV-8 target cell recognition and that HHV-8 envelope protein K8.1 is at least one of the proteins involved.

Anti-apoptotic strategies of lymphotropic viruses

Induction of apoptosis of virus-infected cells is an important host cell defence mechanism. However, some viruses have incorporated genes that encode anti-apoptotic proteins or modulate the expression of cellular regulators of apoptosis. Here, Edgar Meinl and colleagues discuss recent evidence that viral interference with host cell apoptosis leads to enhanced viral replication, and to evasion of cytotoxic T-cell effects.

HIV entry inhibition by the envelope 2 glycoprotein of GB virus C.

Epidemiological studies have revealed an association between GB virus C (GBV-C) long-term viraemia and ameliorated HIV disease progression. We have provided evidence that a single protein of GBV-C, the glycoprotein E2, interferes with early HIV replication steps of both X4- and R5-tropic HIV strains. Preincubation with anti-E2 antibody specifically abrogates the inhibitory effect. Results were confirmed by the in-vitro expression of GBV-C E1/E2 encoding RNA.

Signaling lymphocytic activation molecule (SLAM) regulates T cellular cytotoxicity

Signaling lymphocytic activation molecule (SLAM) is a CD2‐related surface receptor expressed by activated T cells and B cells. SLAM is a self ligand and enhances T cellular proliferation and IFN‐γu2009 production. A defective SLAM associated protein (SAP) causes X‐linked lymphoproliferative syndrome (XLP), a frequently lethal mononucleosis based on the inability to control EBV. We reportthat SLAM augments TCR‐mediated cytotoxicity. In normal CD4+ and CD8+ T cells, SLAM enhanced TCR‐mediated cytotoxicity. In CD4+ and CD8+ Herpesvirus saimiri (H.saimiri) infected T cells, SLAM engagement alone triggered cytotoxicity. Using H.saimiri‐transformed T cells as a model system we found that SLAM‐engagement promotes the release of lytic granules and a CD95‐independent killing that requires extracellular Ca2+, cytoskeletal rearrangements, and signaling mediated by mitogen‐activated protein kinase kinases MEK1/2. SLAM‐enhanced cytotoxicity implies an immunoregulatory function by facilitating the elimination of APC and a role in overcoming infections with pathogens requiring a cytotoxic immune response.

Structural organization of the conserved gene block of Herpesvirus saimiri coding for DNA polymerase, glycoprotein B, and major DNA binding protein

Lymphotropic herpesviruses such as Epstein-Barr virus and Herpesvirus saimiri are commonly grouped as gamma-herpesviruses, although overall genome organization and numerous biological properties are quite different in the viruses. To define the relationship more precisely, we sequenced the Kpnl fragments F (6.5 kb) and C (9.8 kb) of the H.saimiri strain No. 11 genome; these DNA fragments were found to contain the genes coding for equivalents of the major DNA binding protein, a putative glycoprotein transport polypeptide, the glycoprotein B, and the DNA polymerase of herpes simplex virus. This DNA segment represents the longest block of contiguous genes with pronounced sequence homologies between herpesviruses of known DNA primary structure. Comparisons confirmed that the two gamma-herpesviruses are related; the group is, however, even more diverse than the alpha-herpesviruses represented by their prototypes, herpes simplex virus and varicella-zoster virus. H. saimiri DNA is strongly depleted in the dinucleotide CpG, possibly the consequence of de novo methylation of persisting viral DNA in lymphoid cells.

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.

Herpesvirus Saimiri vFLIP Provides an Antiapoptotic Function but Is Not Essential for Viral Replication, Transformation, or Pathogenicity

ABSTRACT Apoptosis of infected cells is an important host defense mechanism, and many viruses have exploited antiapoptotic proteins that interfere with crucial cellular pathways. Viral FLICE inhibitory proteins (vFLIPs) are encoded by rhadinoviruses like herpesvirus saimiri, the related Kaposis sarcoma-associated herpesvirus-human herpesvirus 8 (KSHV/HHV8), and the poxvirus responsible for molluscum contagiosum. The vFLIPs can block the interaction of the death receptor-adapter complex with the cellular effector FLICE (caspase-8), and this prevents the initiation of the downstream caspase cascade. KSHV/HHV8 vFLIP overexpression can confer resistance to T-cell-mediated apoptosis and acts as a tumor progression factor in a murine B-cell lymphoma model. To analyze the function of herpesvirus vFLIPs in the genetic background of the virus and in a model for viral pathogenesis, we deleted the vFLIP gene (open reading frame 71) from the genome of herpesvirus saimiri strain C488. The viral deletion mutant was viable and replicated like the wild-type virus. An antiapoptotic effect could be attributed to the vFLIP gene, but we also show that the vFLIP gene of herpesvirus saimiri is dispensable for viral transformation of T cells in vitro and for pathogenicity in cottontop tamarins in vivo.

The tumor marker Fascin is strongly induced by the Tax oncoprotein of HTLV-1 through NF-κB signals

Oncogenic transformation of CD4(+) T cells by human T-cell lymphotropic virus type 1 (HTLV-1) is understood as the initial step to adult T-cell leukemia/lymphoma, a process that is mainly initiated by perturbation of cellular signaling by the viral Tax oncoprotein, a potent transcriptional regulator. In search of novel biomarkers with relevance to oncogenesis, we identified the tumor marker and actin-bundling protein Fascin (FSCN1) to be specifically and strongly up-regulated in both HTLV-1-transformed and adult T-cell leukemia/lymphoma patient-derived CD4(+) T cells. Fascin is important for migration and metastasis in various types of cancer. Here we report that a direct link can exist between a single viral oncoprotein and Fascin expression, as the viral oncoprotein Tax was sufficient to induce high levels of Fascin. Nuclear factor-κB signals were important for Tax-mediated transcriptional regulation of Fascin in T cells. This suggests that Fascin up-regulation by Tax contributes to the development of HTLV-1-associated pathogenesis.

Susceptibility toHerpesvirus saimiri and antibody development in old and new world monkeys

Herpesvirus saimiri (HVS) induced malignant lymphoma in four cotton-topped (CT) marmosets (S. oedipus). The tumor of a CT marmoset was transplanted to a white-lipped (WL) marmoset (S. nigricollis) which developed malignant lymphoma. The inoculation of HVS and the transplantation of tumor cells did not induce malignant lymphoma in nine adultCallithrix jacchus monkeys and six adultCercopithecus aethiops monkeys. A 32-day-old Callithrix jacchus baby monkey died with malignant lymphoma after infection with HVS. All adult animals of the New World monkey species (S. oedipus, S. nigricollis, Callithrix jacchus) inoculated once with HVS or tumor cells developed equally high titers of neutralizing antibodies against HVS whether they developed a tumor or not. No neutralizing antibodies were detectable in the sera of the Old World monkeys (Cercopithecus aethiops) inoculated with HVS or tumor cells.ZusammenfassungHerpesvirus saimiri (HVS) induzierte in vier cotton topped (CT) Marmosets (S. oedipus) ein malignes Lymphom. Lymphoide Zellen aus dem Tumorgewebe eines CT Marmoset wurden in einen white lipped (WL) Marmoset (S. nigricollis) transplantiert. Das Empfängertier entwickelte ein generalisiertes malignes Lymphom. Die Inoculation von HVS und die Transplantation von Tumorzellen induzierte jedoch keinen Tumor in neun ausgewachsenen Cercopithecus aethiops-Affen. Ein 32 Tage altesCallithrix jacchus-Baby starb an einem malignen Lymphom nach Infektion mit HVS. Alle ausgewachsenen Neuweltaffen (S. oedipus, S. nigricollis, Callithrix jacchus), die einmal mit HVS oder Tumorzellen inoculiert worden waren, bildeten neutralisierende Antikörper gegen HVS in gleicher Titerhöhe, unabhängig davon, ob sie einen Tumor entwickelten oder nicht. In den Seren der Altweltaffen (Cercopithecus aethiops), die mit HVS oder Tumorzellen inoculiert worden waren, konnten keine neutralisierenden Antikörper nachgewiesen werden.

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.