Herpesvirus Saimiri Research Articles

Herpesvirus Saimiri STP-A Oncoprotein Utilizes Src Family Protein Tyrosine Kinase and Tumor Necrosis Factor Receptor-Associated Factors To Elicit Cellular Signal Transduction

The saimiri transforming protein oncogene, called STP-A, of herpesvirus saimiri (HVS) subgroup A is not required for viral replication but is required for lymphoid cell immortalization in culture and lymphoma induction in primates. Here we report that STP-A interacts with cellular tumor necrosis factor receptor-associated factors (TRAF2 and TRAF6) and Src family protein tyrosine kinases (SF-PTKs) in a genetically and functionally separable manner and that each interaction constitutively elicits independent cellular signal transduction. The amino-terminal and central proline-rich motifs of STP-A were responsible for TRAF6 and TRAF2 interactions, respectively, and STP-A and TRAF6 interaction contributed to the majority of NF-kappaB activation, whereas STP-A and TRAF2 interaction played a minor role in NF-kappaB activation. On the other hand, interaction of STP-A with SF-PTKs through its SH2 binding motif effectively elicited AP-1 and NF-AT transcription factor activity. One cellular gene targeted by STP-A is intercellular adhesion molecule 1 (ICAM-1), which participates in a wide range of inflammatory and immune responses. Both TRAF and SF-PTK signal transductions induced by STP-A were required for the marked increase of ICAM-1 expression. These results demonstrate that the viral oncogene STP-A independently targets two vital cellular signaling molecules and that these activities likely contribute to HVS-mediated lymphoid cell immortalization in culture and lymphoma induction in primates.

Functional Characterization and Conformational Analysis of the Herpesvirus saimiri Tip-C484 Protein

Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1–187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.

The Role of Proteinase Inhibitor 9 (PI-9) in Granzyme B/Perforin-Mediated Cytotoxicity Induced by Cytotoxic T Cells in Allogeneic Hematopoietic Stem Cell Transfusion.

The serpin proteinase inhibitor 9 (PI-9) protects cells from serine protease granzyme B (GrB)/perforin-induced apoptosis and cytotoxicity, which is one of the important mechanisms in immunotherapy against tumors. However, the role of PI-9, which specifically inhibits GrB, in tumor immune escape is still under debate. Graft-versus-leukemia (GVL) effect and graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) are the reciprocal aspects the established immunotherapeutic approach in hematopoietic malignancies, and are thought to be caused at least partly through apoptotic molecules GrB and perforin produced by the donor-derived cytotoxic T cells (CTLs) and natural killer cells. However, the role of PI-9 in GVHD and GVL is unknown. In this study we analyzed the role of PI-9 in GVHD and GVL after HSCT through expression of PI-9 relative to GrB and perforin.First, we analyzed the expressions of PI-9 and three apoptosis-related molecules GrB, perforin and Fas ligand by quantitative RT-PCR in leukocytes of five patients with chronic GVHD and six patients without obvious GVHD, from whom a written informed consent was obtained. The level of GrB expression was significantly increased in three of the patients with GVHD and two of them exhibited elevation both in GrB/perforin and FasL. One of the cases with GVHD showed an isolated high expression of FasL. However, significantly, the expression of PI-9 was prominently decreased in all of the cases with GVHD relative to those without GVHD. None of the patients with GVHD who were positive for any of the apoptosis-related molecules relapsed. Two of the six patients without GVHD showed an elevated expression of GrB and perforin. The one of these two patients suffered from relapsed leukemic infiltration into various tissues and the other from herpes zoster reactivation. Thus, the expression of these molecules was thought to reflect the recipients' protective reactions against the intrinsic diseases. We observed a tendency that the ratio of GrB and PI-9 expressions were higher in patients with GVHD.Next, human CTL and natural killer cell (YT-N10) lines were analyzed to check the time course of PI-9 and GrB expressions in vitro. The CTL line had been established by immortalization of normal T cells with herpesvirus saimiri. The stimulation of these cells with phorbol 12-myristate 13 acetate (PMA) resulted in the prior peak of expression of PI-9 within 3 hours followed by the late expression of GrB peaked after 8 hours. The co-stimulation of these cells with IL-2 and IL-12 showed similar results.These results have indicated that the earlier synthesis of PI-9 might be important for escaping autolysis of immunocompetent cells and that the expressions of PI-9 and the apoptosis-related molecules may be controlled in a different manner. The low expression of PI-9 relative to GrB/perforin in patients who received HSCT may indicate that the balance between the expressions of PI-9 and GrB/perforin could monitor the activity of CTLs in patients with post-transplantation GVHD (and possibly with various other immune disorders) and predict the prognosis of these diseases. It could also be correlated with the GVL activity and hence predict the probability of relapses.

381 POSTER HSP27 as a novel target in Non-small cell lung cancer with particular implications for migration and metastasis

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.

Nucleolar trafficking is essential for nuclear export of intronless herpesvirus mRNA

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.

Activation of non-canonical NF-κB pathway mediated by STP-A11, an oncoprotein of Herpesvirus saimiri

Although Saimiri Transforming Protein (STP)-A11, an oncoprotein of Herpesvirus saimiri, has been known to activate NF-κB signaling pathway, the detailed mechanism has not been reported yet. We herein report that STP-A11 activates non-canonical NF-κB pathway, resulting in p100 processing to p52. In addition, translocation of p52 protein (NF-κB2) into the nucleus is observed by the expression of STP-A11. STP-A11-mediated processing of p100 to p52 protein requires proteosome-mediated proteolysis because MG132 treatment clearly blocked p52 production in spite of the expression of STP-A11. Analysis of STP-A11 mutants to activate NF-κB2 pathway discloses the requirement of TRAF6-binding site not Src-binding site for STP-A11-mediated NF-κB2 pathway. Blockage of STP-A11-mediated p52 production using siRNA against p52 enhanced a chemotherapeutic drug-mediated cell death, suggesting that p52 production induced by the expression of STP-A11 would contribute to cellular transformation, which results from a resistance to cell death.

Gene Structure and Expression of Kaposi's Sarcoma-Associated Herpesvirus ORF56, ORF57, ORF58, and ORF59

Though similar to those of herpesvirus saimiri and Epstein-Barr virus (EBV), the Kaposi's sarcoma-associated herpesvirus (KSHV) genome features more splice genes and encodes many genes with bicistronic or polycistronic transcripts. In the present study, the gene structure and expression of KSHV ORF56 (primase), ORF57 (MTA), ORF58 (EBV BMRF2 homologue), and ORF59 (DNA polymerase processivity factor) were analyzed in butyrate-activated KSHV(+) JSC-1 cells. ORF56 was expressed at low abundance as a bicistronic ORF56/57 transcript that utilized the same intron, with two alternative branch points, as ORF57 for its RNA splicing. ORF56 was transcribed from two transcription start sites, nucleotides (nt) 78994 (minor) and 79075 (major), but selected the same poly(A) signal as ORF57 for RNA polyadenylation. The majority of ORF56 and ORF57 transcripts were cleaved at nt 83628, although other nearby cleavage sites were selectable. On the opposite strand of the viral genome, colinear ORF58 and ORF59 were transcribed from different transcription start sites, nt 95821 (major) or 95824 (minor) for ORF58 and nt 96790 (minor) or 96794 (major) for ORF59, but shared overlapping poly(A) signals at nt 94492 and 94488. Two cleavage sites, at nt 94477 and nt 94469, could be equally selected for ORF59 polyadenylation, but only the cleavage site at nt 94469 could be selected for ORF58 polyadenylation without disrupting the ORF58 stop codon immediately upstream. ORF58 was expressed in low abundance as a monocistronic transcript, with a long 5' untranslated region (UTR) but a short 3' UTR, whereas ORF59 was expressed in high abundance as a bicistronic transcript, with a short 5' UTR and a long 3' UTR similar to those of polycistronic ORF60 and ORF62. Both ORF56 and ORF59 are targets of ORF57 and were up-regulated significantly in the presence of ORF57, a posttranscriptional regulator.

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.

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

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.

Functional characterization of the complement control protein homolog of Herpesvirus saimiri: R118 is critical for factor I cofactor activities

Functional characterization of the complement control protein homolog of Herpesvirus saimiri: R118 is critical for factor I cofactor activities

Functional Characterization of the Complement Control Protein Homolog of Herpesvirus Saimiri: ARG-118 IS CRITICAL FOR FACTOR I COFACTOR ACTIVITIES

Herpesvirus saimiri (HVS) is a lymphotropic virus that causes T-cell lymphomas in New World primates. It encodes a structural homolog of complement control proteins named complement control protein homolog (CCPH). Previously, CCPH has been shown to inhibit C3d deposition on target cells exposed to complement. Here we have studied the mechanism by which it inactivates complement. We have expressed the soluble form of CCPH in Escherichia coli, purified to homogeneity and compared its activity to vaccinia virus complement control protein (VCP) and human complement regulators factor H and soluble complement receptor 1. The expressed soluble form of CCPH bound to C3b (KD = 19.2 microm) as well as to C4b (KD = 0.8 microm) and accelerated the decay of the classical/lectin as well as alternative pathway C3-convertases. In addition, it also served as factor I cofactor and supported factor I-mediated inactivation of both C3b and C4b. Time course analysis indicated that although its rate of inactivation of C4b is comparable with VCP, it is 14-fold more potent than VCP in inactivating C3b. Site-directed mutagenesis revealed that Arg-118, which corresponds to Lys-120 of variola virus complement regulator SPICE (a residue critical for its enhanced C3b cofactor activity), contributes significantly in enhancing this activity. Thus, our data indicate that HVS encodes a potent complement inhibitor that allows HVS to evade the host complement attack.

Interleukin-26, An Epitheliotropic T-Cell Cytokine

Interleukin-26 (IL-26) belongs to the family of cellular cytokines which share sequence homology with IL-10, however, without functional conservation. The il-26 gene is situated on the human chromosome 12q15 in close neighbourhood to the genes for the related T-cellular cytokines interferon-γ (IFN-γ) and IL-22. il-26 was discovered due to its overexpression in T cells after growth transformation with herpesvirus saimiri. IL-26 is produced by activated T cells, forms homodimers, and acts on epithelial target cells such as colon carcinoma cells or keratinocytes. Presumably due to its positive net charge, IL-26 adheres to glycosaminoglycans on cell surfaces. Stimulation with IL-26 results in the phosphorylation and activation of the transcription factors STAT1 and STAT3 and in the induction of CD54 surface expression and secretion of cytokines such as IL-8 and IL-10. These effects are mediated by an IL-26-specific receptor complex consisting of IL-20R1 and IL-10R2. Thus, IL-26 appears to be a rather proinflammatory and cell-surface associated cytokine linking local T-cell activation with epithelial functions.

StpC-based gene therapy targeting latent reservoirs of HIV-1

The ability of HIV-1 to form latent reservoirs presents a major obstacle to eradication. One approach to elimination of the latent reservoir is induction therapy, whereby cells harboring latent virus are activated and therefore initiate virus replication. We have constructed a lentiviral vector encoding Herpesvirus saimiri subgroup C saimiri transformation-associated protein (StpC), which has been shown to modulate HIV-1 replication, under the control of a cytomegalovirus promoter in order to determine the ability of StpC to upregulate latent HIV-1. We have included a suicide gene, herpes simplex virus thymidine kinase (TK), under the control of the HIV-1 long terminal repeat (LTR) promoter. We hypothesized that upon StpC expression in latently infected cells induction of virus replication and subsequent production of viral transactivators of the LTR will activate expression of the tk gene, sensitizing the cells to the nucleoside analogue ganciclovir (GCV). Transduction of the latently infected cell line J1.1 resulted in increased virus replication. In the presence of GCV transduced cells exhibited decreased HIV-1 replication, inhibition of cell proliferation, and increased apoptosis. This prototype vector serves as a proof of concept of the utility of gene-based induction agents and suicide genes as a new method for targeting reservoirs of latent HIV-1.

CD4 + lymphocytes require platelets for adhesion to immobilized fibronectin in flow: Role of β 1 (CD29)-, β 2 (CD18)-related integrins and non-integrin receptors

The role of platelets in T-lymphocytes adhesion is not clear yet. Herpesvirus saimiri (HVS)-infected CD4 + T-lymphocytes were placed into polystyrene plates pre-coated with fibronectin. The adherent T-cells were enumerated by image analysis. Under static condition, 38 ± 10 cells/mm 2 adhered and addition of gel-filtered platelets (GFP) and PMA enhanced cell adhesion 4.3- and 4.1-fold. Using PMA plus GFP 11.9-fold enhancement in cell adhesion was achieved. In contrast, under flow (200 s −1), neither basal adhesion nor following separate addition of PMA or GFP was observed, whereas combined addition of PMA and GFP induced noticeable adhesion (34 cells/mm 2). The adhesion was inhibited by blockade of α 5-integrin (CD49e, 87%), β 2-integrin (CD18, 78%), CD40L (60%), PSGL-1 (CD162, 60%), and CD40L plus PSGL-1 (83%). Thus, activated platelets promote activated T-cell adhesion to fibronectin under flow via integrins (α 5β 1, and α Lβ 2), CD40–CD40L and P-selectin–PSGL-1 mediated interactions.

H. saimiri tyrosine-kinase interacting protein inhibits Tat function: A prototypic strategy for restricting HIV-1-induced cytopathic effects in immune cells

Herpesvirus saimiri (HVS)-transformed human T cells become permissive for X4 and R5 strains of human immunodeficiency virus type 1 (HIV-1), evidence that HVS-encoded proteins associated with T cell transformation enhance HIV-1 replication. Analyzing the contribution of transformation-associated bicistronic HVS open reading frames (ORF) to HIV-1 replication revealed expression of the second ORF saimiri transformation-associated protein type C (StpC) conferred the permissive phenotype to T cells. In contrast, expression of the first HVS ORF tyrosine-kinase interacting protein (Tip) in the absence of StpC enhanced restriction of HIV-1 replication in T cell lines and peripheral blood mononuclear cells. Understanding the mechanism whereby Tip enhanced restriction of HIV-1 replication may uncover unique pathways that could be targeted therapeutically. Here we report that Tip restricts HIV-1 replication in a monocyte-derived cell line and restricts reactivation of replication of HIV-1 in a T cell line harboring provirus. In this report, we begin to unravel the molecular underpinnings of Tip-mediated restriction. Tip mediates both lymphocyte-cell-specific kinase (Lck)-dependent and -independent effects on HIV-1 replication. We also provide evidence that Tip-mediated restriction is in part due to inhibition of Tat transactivation of the HIV-1 long terminal repeat (LTR). Expression of Tip in T cells increased activation of Stat1 and Stat3, as well as activation of protein kinase RNA-dependent (PKR/p68) and interferon-γ production. Taken together, these results provide evidence that Tip restricts HIV-1 replication and reactivation by inhibiting HIV-1 transcription while inducing an intercellular antiviral state. We propose that genetically engineered vectors driving Tip expression could provide a prototypic strategy for restricting HIV-1 replication and reactivation in diverse cell lineages.

Regulation of intracellular signalling by the terminal membrane proteins of members of the Gammaherpesvirinae

The human gamma(1)-herpesvirus Epstein-Barr virus (EBV) and the gamma(2)-herpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV), rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) all contain genes located adjacent to the terminal-repeat region of their genomes, encoding membrane proteins involved in signal transduction. Designated 'terminal membrane proteins' (TMPs) because of their localization in the viral genome, they interact with a variety of cellular signalling molecules, such as non-receptor protein tyrosine kinases, tumour-necrosis factor receptor-associated factors, Ras and Janus kinase (JAK), thereby initiating further downstream signalling cascades, such as the MAPK, PI3K/Akt, NF-kappaB and JAK/STAT pathways. In the case of TMPs expressed during latent persistence of EBV and HVS (LMP1, LMP2A, Stp and Tip), their modulation of intracellular signalling pathways has been linked to the provision of survival signals to latently infected cells and, hence, a contribution to occasional cellular transformation. In contrast, activation of similar pathways by TMPs of KSHV (K1 and K15) and RRV (R1), expressed during lytic replication, may extend the lifespan of virus-producing cells, alter their migration and/or modulate antiviral immune responses. Whether R1 and K1 contribute to the oncogenic properties of KSHV and RRV has not been established satisfactorily, despite their transforming qualities in experimental settings.

Conformational analysis of the Tip protein from Herpesvirus saimiri with hydrogen exchange and mass spectrometry

Conformational analysis of the Tip protein from Herpesvirus saimiri with hydrogen exchange and mass spectrometry

3. Cytokines and chemokines

Cytokines and chemokines are secreted proteins with growth, differentiation, and activation functions that regulate the nature of immune responses. Cytokines are involved in nearly every facet of immunity and inflammation, from induction of the innate immune response to the generation of cytotoxic T cells and the development of antibodies by the humoral immune system. The combination of cytokines that are produced in response to an immune insult determines which arm of the immune system will be activated. For this update, recent advances in our understanding of cytokines will be discussed, which includes the IL-10, IL-17, and IL-27 families.

Herpesvirus Saimiri-Based Gene Delivery Vectors

Herpesviruses possess a number of characteristics which make them promising gene delivery vectors. These include their capacity to package large amounts of heterologous DNA and an ability to establish persistent, lifelong infections, where the viral genome remains as a circular non-integrated episome. Herpesvirus saimiri (HVS) is the prototype gamma-2 herpesvirus and is currently being developed as a potential gene delivery vector. In addition to the above properties, HVS-based vectors have the ability to infect a wide range of human cell lines and primary cultures with high efficiencies. Moreover, upon infection the viral genome persists as high copy number, circular, non-integrated episomes which segregate to progeny cells upon division. This allows the HVS-based vector to stably transduce a dividing cell population and provide sustained heterologous gene expression. As such, it offers the characteristics of an artificial chromosome combined with a highly efficient delivery system. This review aims to describe the assessment of HVS-based vectors in both in vitro and in vivo studies, highlighting new developments and possible applications for the treatment of genetic diseases.

Herpesvirus saimiri (HVS)-transformed T-cell lines: A method to study mucosal T cells in inflammatory bowel disease

Herpesvirus saimiri (HVS)-transformed T-cell lines: A method to study mucosal T cells in inflammatory bowel disease