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.

Lck protein tyrosine kinase is a key regulator of T-cell activation and a target for signal intervention by Herpesvirus saimiri and other viral gene products.

Protein tyrosine kinases (PTKs) are critically involved in signaling pathways that regulate cell growth, differentiation, activation, and transformation. It is not surprising, therefore, that viruses acquire effector molecules targeting these kinases to ensure their own replication and/or persistence. This review summarizes our current knowledge on Lck, a member of the Src family of PTK, and its viral interaction partners. Lck plays a key role in T lymphocyte activation and differentiation. It is associated with a variety of cell surface receptors and is critical for signal transduction from the T-cell antigen receptor (TCR). Consequently, Lck is targeted by regulatory proteins of T-lymphotropic viruses, especially by the Herpesvirus saimiri (HVS) tyrosine kinase interacting protein (Tip). This oncoprotein physically interacts with Lck in HVS transformed T cells and has an impact on its catalytic activity. However, while Tip inhibits Lck activity in stably expressing cell lines, opposite effects were observed in several in vitro systems. At least in part, this complex situation may be related to the bipartite nature of the interaction surface of the two proteins. Studies on the interrelationships between Lck and its viral partners contribute to the understanding of the mechanisms of T-cell growth regulation, in general, and of viral pathogenicity in particular. In addition, understanding the regulation of Lck activity by viral proteins may serve as a basis for the development of new drugs capable of modifying Lck activity in different pathological situations.

Herpesvirus saimiri as a model for gammaherpesvirus oncogenesis.

Herpesvirus saimiri (HVS) causes T-lymphoproliferative dis-$borders in several New World and Old World primate species and in certain rabbits.In vitro infection leads to permanent growth of primary T cells of primate and human origins. The transformation-relevant proteins of HVS interact with cellular proto-oncoproteins which results in cell growth transformation. In addition, virus-encoded cellular homologues may contribute to transformation or persistence of HVS by altering cellular signal transduction and deregulating cell growth control. Because of the presence of a permissive cell culture system and in vitro Land in vivo transformation assays, HVS provides a unique opportunity to investigate the mechanisms of cancer induction by oncogenic herpesviruses.

Herpesvirus ateles gene product Tio interacts with nonreceptor protein tyrosine kinases.

Herpesvirus ateles is a gamma-2-herpesvirus which naturally infects spider monkeys (Ateles spp.) and causes malignant lymphoproliferative disorders in various other New World primates. The genomic sequence of herpesvirus ateles strain 73 revealed a close relationship to herpesvirus saimiri, with a high degree of variability within the left terminus of the coding region. A spliced mRNA transcribed from this region was detected in New World monkey T-cell lines transformed by herpesvirus ateles in vitro or derived from T cells of infected Saguinus oedipus. The encoded viral protein, termed Tio, shows restricted homology to the oncoprotein StpC and to the tyrosine kinase-interacting protein Tip, two gene products responsible for the T-cell-transforming and oncogenic phenotype of herpesvirus saimiri group C strains. Tio was detectable in lysates of the transformed T lymphocytes. Dimer formation was observed after expression of recombinant Tio. After cotransfection, Tio was phosphorylated in vivo by the protein tyrosine kinases Lck and Src and less efficiently by Fyn. Stable complexes of these Src family kinases with the viral protein were detected in lysates of the transfected cells. Binding analyses indicated a direct interaction of Tio with the SH3 domains of Lyn, Hck, Lck, Src, Fyn, and Yes. In addition, tyrosine-phosphorylated Tio bound to the SH2 domains of Lck, Src, or Fyn. Thus, herpesvirus ateles-encoded Tio may contribute to viral T-cell transformation by influencing the function of Src family kinases.

Dominant clones in immortalized T-cell lines from rheumatoid arthritis synovial membranes.

Transformation of human T cells by herpesvirus saimiri allows the production of an unlimited number of T cells which express a functional T-cell receptor. In this study we transformed four T-cell lines derived from rheumatoid arthritis synovial membranes. The transformed T cells were mainly CD4+ and expressed the phenotype of activated T cells. They were grown for more than 1 year in the absence of mitogen or feeder cells, and three of them could be maintained without exogenous IL-2. The presence of viral DNA in the transformed cells was shown by in situ hybridization with a probe from the H-DNA region of the virus. No infectious virus could be recovered from the transformed cells. The relative proportion of the 24 different Vbeta families between the four transformed lines showed variations that increased with time. In the two T-cell lines transformed at an early stage of culture, the Vbeta2 family was maintained at about 10%. The dominant Vbeta2 clones that previously have been characterized in the patient were found in all transformed T-cell lines. We have thus shown the feasibility of obtaining transformed T cells from synovial membranes. They contain the dominant clones that are considered of potential importance for the disease, permitting further functional studies.

Functional phenotype of transformed human alphabeta and gammadelta T cells determined by different subgroup C strains of herpesvirus Saimiri.

Based on sequence divergence in the transformation-relevant region, herpesvirus saimiri strains are classified into three subgroups. Only members of subgroup C transform human T lymphocytes to continuous interleukin-2-dependent growth in culture. In this study, human cord blood T cells were immortalized by using different subgroup C strains (C488, C484, and C139). The resulting T-cell lines represented different types of T-cell clones. They were either CD4+ or CD8+ and expressed either the alphabeta or the gammadelta type of T-cell receptors. If transformed by the same virus strain, alphabeta and gammadelta clones were similar with respect to viral persistence, virus gene expression, proliferation, and Th1-type cytokine production. However, major differences were observed in T cells immortalized by different subgroup C strains. Strain C139 persisted at low copy number, compared to the high copy number of prototype C488. The transformation-associated genes stpC and tip of strain C488 were strongly induced after T-cell stimulation. The homologous genes of strain C139 were only weakly expressed and not induced after activation. After CD2 ligation, the C488-transformed T cells produced interleukin-2, whereas the C139-transformed cells did not. Correspondingly, the C139-transformed T cells were less sensitive to cyclosporin A. Sequence comparison from different subgroup C strains revealed a variability of the stpC/tip promoter region and of the Lck-binding viral protein Tip. Thus, closely related subgroup C strains of herpesvirus saimiri cause major differences in the functional phenotype of growth-transformed human T cells.

Regulation of the herpesvirus saimiri oncogene stpC, similar to that of T-cell activation genes, in growth-transformed human T lymphocytes.

Herpesvirus saimiri strain C488, a T-cell tumor virus of New World primates, transforms human T lymphocytes to stable interleukin-2-dependent growth without need for further stimulation by antigen or mitogen. The transformed cell lines show the phenotype of activated mature T cells and retain many essential features of the primary parental cells, e.g., antigen specificity. In contrast to transformed New World monkey T cells, the human lines do not support lytic growth of the virus, even after chemical stimulation. Here we show that many viral genes remain silent during episomal persistence. However, the viral oncogene stpC is predominantly transcribed and translated to a stable cytoplasmic protein of 20 kDa that is heterogeneously expressed in individual cells. This 1.7-kb mRNA is bicistronic, encoding also Tip, a viral protein interacting with the T-cell-specific tyrosine kinase Lck. stpC/tip transcripts are heavily induced upon stimulation by mitogen or phorbol ester. Block of protein synthesis does not abolish transcription: treatment with cycloheximide greatly induces stpC/tip mRNA levels. Thus, this gene complex is regulated similarly to early T-cell activation genes. Constitutive and induced expression engage different transcription start sites. The T-cell regulation of the viral genes stpC and tip may contribute to the T-cell tropism of growth transformation by herpesvirus saimiri.

Herpesvirus saimiri immortalized gamma delta T cell line activated by IL-12.

IL-12 is a novel heterodimeric cytokine important for the regulation and differentiation of lymphocytes and NK cells. Like other cytokines, IL-12 mediates its biologic activity through high-affinity receptors expressed on responsive cells. To date, a large number of receptors for IL-12 have been found only on PBMC following activation with PHA or IL-2. To gain further knowledge of the IL-12R complex and the IL-12 signal transduction pathway in cytotoxic T cells, we studied a number of human T cell lines that had been transformed to permanent growth with Herpesvirus saimiri, an oncogenic virus of nonhuman primates. This paper reports the expression of IL-12R on a human gamma delta T cell line that responds to IL-12 with enhanced cytolytic activity and increased expression of cytolytic effector molecules granzyme B and perforin. Using these T cells as a model of IL-12 signal transduction, we confirmed that these events involve members of the Janus kinase family of nonreceptor tyrosine kinases JAK2, TYK2, and signal transducer and activator of transcription 4.

A Herpes saimiri oncogene causing peripheral T-cell lymphoma in transgenic mice.

Herpesvirus saimiri is an oncogenic virus causing rapid T-cell lymphomas in New World primates and rabbits. Deletion analysis of one strain of H saimiri has indicated an open reading frame, StpA, necessary for oncongenicity in monkeys. We have investigated the function of StpA in tumor induction by the generation of transgenic mice. Expression of two different constructs caused the development of peripheral lymphomas. The infiltrating cells were of T-cell origin, expressing mainly the CD4 phenotype and restricted sets of V beta chains. Thus, StpA is not only necessary for the oncogenicity of Herpesvirus saimiri, but is also sufficient for the induction of peripheral pleomorphic T-cell lymphomas.

[Absence of herpesvirus saimiri STPC oncogene in salivary gland tumors and epithelial thymus tumors]. / Fehlen des Herpesvirus saimiri Onkogens STPC in Speicheldrüsen-tumoren und epithelialen Thymustumoren.

AIM AND METHODS: The oncoprotein STP-C-488 induces salivary gland and thymic epithelial tumours when expressed as a transgene in mice (MURPHY et al. 1994). Given the enigmatic tumorigenesis of corresponding tumours in humans, we now investigated genomic DNA and RNA from 11 thymomas, 5 pleomorphic adenomas and control autopsy material (n = 8) for the occurrence of the STP-C-488 sequences by Southern-blotting, Northern-blotting and PCR. RESULTS: All tumor samples and control tissues were negative for the STP-C-488 in Southern-blot and Northern-blot-hybridization. PCR analyses did not reveal amplification products of the length expected for STP-C-488. However, a PCR fragment of a different size was found in 50% of the thymomas and pleomorphic adenomas, but in only one of 8 controls. The sequence of this PCR product revealed local homologies with various herpesviruses. CONCLUSION: The oncoprotein STP-C-488 is not involved in the tumorigenesis of human thymomas and salivary gland tumours. Whether the novel sequences amplified preferentially from these tumours play a role in pathogenesis needs further investigation.

Identification of Lck-binding elements in tip of herpesvirus saimiri.

A protein called Tip (tyrosine kinase interacting protein) of herpesvirus saimiri associates with Lck in virus-transformed human T cells and is an in vitro substrate for Lck kinase. Mutational analyses of a GST-Tip fusion protein revealed that binding to Lck requires putative SH3 binding sequences and a sequence homologous to the carboxyl terminus of Src-related kinases. These sequences are referred to as SH3-Binding (SH3B) and C-terminal Src-related Kinase Homology (CSKH) elements. Peptide fragments as short as 37 amino acids containing both SH3B and CSKH elements were sufficient to form a stable complex with Lck in vitro. Furthermore, these same sequences of Tip were necessary for in vivo association with Lck when Tip and Lck were expressed transiently in COS-1 cells or stably in Rat-1 cell lines. These results demonstrate that the CSKH element of Tip participates in the binding of sequences within Lck. Tip of herpesvirus saimiri has apparently acquired such CSKH and SH3B elements for the purpose of targeting cellular protein kinases. The interaction of Tip with Lck may influence Lck kinase activity or its binding to other cellular proteins and thereby alter Lck function in T cells infected by h. saimiri.

The product of the Herpesvirus saimiri open reading frame 1 (tip) interacts with T cell-specific kinase p56lck in transformed cells.

Subgroup C strains of Herpesvirus saimiri, a leukemogenic virus of non-human primates, transform human T cells to permanent growth in culture. These cell retain their antigen specificity, and they are becoming widely used as a model for activated human T cells. Though a variety of human cell types can be infected by H. saimiri, transformation appears to be specific for CD4+ and CD8+ T cells. Our investigation of early signaling events in H. saimiri-transformed T cells revealed a novel 40-kDa phosphoprotein complexed with the T cell-specific tyrosine protein kinase p56lck. This protein, termed Tip (tyrosine kinase interacting protein), is identified as a viral protein encoded by the open reading frame 1 (ORF1). In the transformed cells Tip is expressed together with the gene product of ORF2, the viral oncoprotein StpC, which acts on epithelial cells. The H. saimiri genome has 75 ORFs, but only ORF1 and ORF2 are transcribed in transformed human cells. Tip is phosphorylated on tyrosine in cell-free systems containing Lck, indicating that the viral protein is a substrate for this T cell-specific kinase. Alteration of T cell signaling pathways by Tip may be the second event complementing the action of StpC in a new mechanism of T cell transformation.

Epithelial tumours induced by a herpesvirus oncogene in transgenic mice.

To investigate the role of herpesviral genes in tumourigenesis, transgenic mice were generated expressing STP-C, a transformation associated protein of the lymphoma inducing herpesvirus saimiri. Epithelial tumours developed in the salivary gland, pancreas, thymus and liver of transgenic mice within the first weeks of life. Thus, the target cells for tumour formation in the transgenic mice were surprisingly different from those of the herpesvirus from which the oncogene was derived. Our results identify STP-C as a herpesvirus oncogene sufficient for tumour induction without the cooperation of other viral gene products. Furthermore, the results demonstrate pleiotropic transforming capabilities of the STP-C oncogene and suggest that the specificity of lymphoma induction by the virus is determined by factors other than the oncogene itself.

Immortalization of human T cell clones by Herpesvirus saimiri. Signal transduction analysis reveals functional CD3, CD4, and IL-2 receptors.

Investigation of human activated T cells has been complicated by the need for periodic restimulation with Ag/mitogen and accessory cells and by the limited life span of most human T cell clones. To overcome these problems, we have transformed established human T cell clones to permanent growth with Herpesvirus saimiri, a lymphoma-inducing virus of nonhuman primates. Three human CD4+ T cell clones were investigated in detail. They have been growing in the presence of exogenous IL-2 but without restimulation with mitogen or feeder cells for more than 11 mo with doubling times between 2 and 4 days. In contrast, their nontransformed parent clones needed to be restimulated with PHA and feeder cells every 14 to 21 days. To compare responses of H. saimiri-transformed clones with those of their parent clones, we stimulated the cells with IL-2 or with anti-CD3 and/or anti-CD4 mAb with and without cross-linking on the cell surface. Transformed and nontransformed T cell clones were strikingly similar in parameters of early signal transduction, namely, tyrosine phosphorylation and mobilization of calcium. Ligation of their TcR/CD3 complexes by mAb or by Ag in the presence of autologous accessory cells increased the proliferation and the secretion of IFN-gamma. Taken together, we have shown that human T cell clones immortalized with H. saimiri express functional CD3, CD4, and IL-2R. They constitute a simple, stable, reproducible and accessory cell-free model system for the investigation of signal transduction events in activated human T cells.

Herpesvirus saimiri transformed human T cell lines: a permissive system for human immunodeficiency viruses.

Human peripheral blood T lymphocytes are readily transformed to continuous growth by Herpesvirus saimiri subgroup C strains. The immortalized cells express the phenotype of mature activated T cells and bear either CD4 or CD8 surface markers. Here we report that Herpesvirus saimiri transformed CD4+ cell lines are highly susceptible to infection with human immunodeficiency viruses types 1 and 2. The prototype viruses HIV-1IIIB and HIV-2ROD replicated rapidly and caused cell death within 14 days. These cell lines did also support growth of a poorly replicating HIV-2 strain (HIV-2NEP) and of primary clinical isolates. Thus H. saimiri transformed T cells represent a new system for HIV propagation and isolation, especially for HIV-2 strains with restricted cell tropism. These cells should be considered as an alternative approach in cases where conventional attempts fail.

Primary structure of the herpesvirus saimiri genome.

This report describes the complete nucleotide sequence of the genome of herpesvirus saimiri, the prototype of gammaherpesvirus subgroup 2 (rhadinoviruses). The unique low-G + C-content DNA region has 112,930 bp with an average base composition of 34.5% G + C and is flanked by about 35 noncoding high-G + C-content DNA repeats of 1,444 bp (70.8% G + C) in tandem orientation. We identified 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes. The genes are closely arranged, with only a few regions of sizable noncoding sequences. For 60 of the predicted proteins, homologous sequences are found in other herpesviruses. Genes conserved between herpesvirus saimiri and Epstein-Barr virus (gammaherpesvirus subgroup 1) show that their genomes are generally collinear, although conserved gene blocks are separated by unique genes that appear to determine the particular phenotype of these viruses. Several deduced protein sequences of herpesvirus saimiri without counterparts in most of the other sequenced herpesviruses exhibited significant homology with cellular proteins of known function. These include thymidylate synthase, dihydrofolate reductase, complement control proteins, the cell surface antigen CD59, cyclins, and G protein-coupled receptors. Searching for functional protein motifs revealed that the virus may encode a cytosine-specific methylase and a tyrosine-specific protein kinase. Several herpesvirus saimiri genes are potential candidates to cooperate with the gene for saimiri transformation-associated protein of subgroup A (STP-A) in T-lymphocyte growth stimulation.

Stable growth transformation of human T lymphocytes by herpesvirus saimiri.

Herpesvirus saimiri induces T-cell lymphomas in various species of New World monkeys and in rabbits, and it is able to immortalize monkey T lymphocytes in vitro. Sequences responsible for these effects have been localized to a region of the genome that varies significantly among the virus subgroups A, B, and C. We now report that infection of human blood lymphocytes and thymocytes with strains of subgroup C, in contrast to viruses of the other subgroups, yields continuously proliferating T-cell lines with the phenotype of mature CD4- or CD8-positive cells. Infection with strains of Herpes-virus saimiri subgroup C can thus be used to generate human T-cell lines for a variety of immunological and developmental studies.

Identification of transforming genes of subgroup A and C strains of Herpesvirus saimiri.

Herpesvirus saimiri is an oncogenic herpesvirus that induces rapidly progressing lymphomas in New World primates. Using retrovirus vectors for gene transfer, specific open reading frames of H. saimiri were tested for their ability to transform rodent cells in culture. One open reading frame, designated STP-C488 (for saimiri-transformation-associated protein of the subgroup C strain 488), phenotypically transformed Rat-1 cells, resulting in formation of foci, growth at reduced serum concentration, and growth to higher cell densities. Cells transformed by STP-C488 formed invasive tumors in nude mice. The STP-A11 reading frame of strain 11 (subgroup A) was much less potent in its transforming ability than STP-C488. These results demonstrate the oncogene nature of these two open reading frames and provide a means for studying their transforming functions independent of the rest of the H. saimiri genome.

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.