Activation of the Lck tyrosine protein kinase by the Herpesvirus saimiri tip protein involves two binding interactions.

The Tip protein of Herpesvirus saimiri strain 484C binds to and activates the Lck tyrosine protein kinase. Two sequences in the Tip protein were previously shown to be involved in binding to Lck. A proline-rich region, residues 132-141, binds to the SH3 domain of the Lck protein. We show here that the other Lck-binding domain, residues 104-113, binds to the carboxyl-terminal half of Lck and that this binding does not require the Lck SH3 domain. Mutated Tip containing only one functional Lck-binding domain can bind stably to Lck, although not as strongly as wild-type Tip. Interaction of Tip with Lck through either Lck-binding domain increases the activity of Lck in vivo. Simultaneous binding of both domains is required for maximal activation of Lck. The transient expression of Tip in T cells was found to stimulate both Stat3-dependent and NF-AT-dependent transcription. Mutant forms of Tip lacking one or the other of the two Lck-binding domains retained the ability to stimulate Stat3-dependent transcription. Tip lacking the proline-rich Lck-binding domain exhibited almost wild-type activity in this assay. In contrast, ablation of either Lck-binding domain abolished the ability of Tip to stimulate NF-AT-dependent transcription. Full biological activity of Tip, therefore, appears to require both Lck-binding domains.

Activation of the lck tyrosine-protein kinase by the binding of the tip protein of herpesvirus saimiri in the absence of regulatory tyrosine phosphorylation.

The Tip protein of herpesvirus saimiri 484 binds to the Lck tyrosine-protein kinase at two sites and activates it dramatically. Lck has been shown previously to be activated by either phosphorylation of Tyr394 or dephosphorylation of Tyr505. We examined here whether a change in the phosphorylation of either site was required for the activation of Lck by Tip. Remarkably, mutation of both regulatory sites of tyrosine phosphorylation did not prevent activation of Lck by Tip either in vivo or in a cell free in vitro system. Tip therefore appears to be able to activate Lck through an induced conformational change that does not necessarily involve altered phosphorylation of the kinase. Tip may represent the prototype of a novel type of regulator of tyrosine-protein kinases.

Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells.

Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability.

The Lck binding domain of herpesvirus saimiri tip-484 constitutively activates Lck and STAT3 in T cells.

Constitutive activation of signal transducers and activators of transcription (STATs) has been associated with oncogenesis. Previously, a protein required for T-cell transformation by the DNA tumor virus herpesvirus saimiri (HVS) strain 484, designated tyrosine kinase-interacting protein (Tip-484), was shown to interact with and dramatically upregulate the activity of the STATs in an Lck-dependent manner. The minimal region of Tip-484 responsible for binding Lck was defined as a 10-residue C-terminal Src-related kinase homology domain, an 18-amino-acid spacer, and a 10-residue potential SH3 binding domain. This region is termed the LBD (for Lck binding domain). The present data show that only the LBD of Tip-484 is needed to activate Lck in vitro and in vivo. Finally, the LBD was shown to form a complex with STAT3 in vitro, and expression of the LBD in T cells led to STAT3 activation equal to that of full-length Tip-484. These studies demonstrate that the 48-amino-acid LBD of Tip-484 can perform as effectively as the full-length protein in vitro and in vivo.

The Src-family kinase Lck can induce STAT3 phosphorylation and DNA binding activity.

Constitutive activation of the Src-family kinase Lck has been shown to lead to transformation. Constitutive activation of the STAT pathway of transcription factors has also been shown to be involved in transformation. An oncogenic form of the prototypical member of the Src-family, v-Src, has been shown to activate STAT3, and this activation is required for v-Src's transforming ability. To investigate whether Lck could directly activate STAT3, a baculovirus expression system was utilised. When Lck and STAT3 were coexpressed, STAT3 was found to have enhanced tyrosine phosphorylation and DNA binding activity. This finding was confirmed with experiments where exogenous Lck was added to baculovirus produced STAT3. Moreover, the activation of STAT3 by exogenous Lck could be attenuated by the Lck-specific inhibitor PP1. In addition, mammalian cells stably expressing a constitutively activated form of Lck were shown to have activated STAT3. These data provide strong evidence that, like v-Src, Lck can also directly activate STAT3, which contributes to the transformation process.

In vitro antiviral drug sensitivity of the Kaposi's sarcoma-associated herpesvirus.

OBJECTIVE: Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, has been implicated as the causative agent of Kaposi's sarcoma. Retrospective studies show that the risk of development of Kaposi's sarcoma is significantly lower in AIDS patients who received ganciclovir or phosphonoformic acid (PFA) therapy. Therefore, in vitro antiviral drug sensitivity of KSHV was studied. METHODS: The KSHV genome is a latent episome in lymphoma cells such as the BCBL-1 cell line. Lytic KSHV DNA synthesis is induced by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate in BCBL-1 cells; this system was used to evaluate the effects of antiviral drugs on KSHV DNA synthesis. RESULTS: Linear (lytic) KSHV DNA synthesis and virus secretion was inhibited in BCBL-1 cell cultures by cidofovir (median inhibitory concentration, 0.05 microM), ganciclovir (5.1 microM) and PFA (97 microM), and by aciclovir (75 microM). Prolonged incubation of BCBL-1 cells with antiviral drugs had no effect on episomal KSHV DNA synthesis. CONCLUSIONS: The antiviral drug assay developed shows that KSHV is very sensitive to cidofovir, moderately sensitive to ganciclovir and PFA, and weakly sensitive to aciclovir. Therefore, low doses of cidofovir, or high doses of PFA or ganciclovir could suppress clinical reactivation of KSHV. Antiviral drugs did not inhibit episomal virus DNA synthesis, suggesting that the latent form of viral DNA is replicated by host DNA polymerases. Consequently, no benefit can be expected from antiviral drugs in KSHV-positive B-cell lymphomas or during latency.

Activation of STAT transcription factors by herpesvirus Saimiri Tip-484 requires p56lck.

Signal transducers and activators of transcription (STATs) relay signals from activated cell surface receptors directly to the nucleus. Previously, a protein required for T-cell transformation by the DNA tumor virus herpesvirus saimiri (HVS) and designated tyrosine kinase interacting protein (Tip-484) was shown to interact with and dramatically upregulate the activity of p56lck. p56lck is a nonreceptor tyrosine kinase that is essential for signaling by the T-cell receptor and also interacts with the CD4, CD8, and interleukin-2 receptors. The present data show activation of STAT1 and -3 by Tip-484. STAT1 and -3 were also found to complex with glutathione S-transferase-Tip-484 only in the presence of p56lck, and STAT3 was shown to be phosphorylated by the Tip-484-p56lck multiprotein complex in vitro. Infection of T cells with HVS or expression of recombinant Tip-484 significantly increased the DNA-binding activity of the STAT1 and STAT3 transcription factors in nuclear extracts and also increased the phosphorylation of STAT3 in vivo. This is the first report of STAT activation by a DNA tumor virus protein. Moreover, these studies demonstrate that p56lck is required for STAT activation by Tip-484.

Herpesvirus saimiri Tip-484 membrane protein markedly increases p56lck activity in T cells.

Herpesvirus saimiri (HVS) is a T-cell-specific transforming and oncogenic virus. A protein encoded by HVS known as Tip-484 (for tyrosine kinase interacting protein from HVS strain 484) is required for this transformation. Tip-484 binds specifically to the nonreceptor protein tyrosine kinase p56lck. By transfecting Tip-484 into T cells, we now show that this interaction leads to a several hundred-fold increase in the kinase activity of p56lck. Tip-484 is part of a protein complex which is dependent on the presence of p56lck and is phosphorylated. We also show that two of the complexed proteins represent two phosphorylated forms of Tip-484. Furthermore, the p56lck kinase activity in HVS-infected human peripheral blood T lymphocytes was at least ninefold higher than that in noninfected control cells and significantly decreased in cells infected with a Tip-484 deletion mutant virus. Finally, we report that Tip-484 is required for oncogenesis in rabbits by the survival of rabbits inoculated with Tip-484 deletion mutant HVS. The data demonstrate dramatic stimulation of the signaling pathway of p56lck. This effect can contribute to the molecular mechanisms that lead to sustained autocrine secretion of growth factors, permanent T-cell growth, and ultimately lymphocytic tumor formation.

Identification of a herpesvirus Saimiri cis-acting DNA fragment that permits stable replication of episomes in transformed T cells.

Herpesvirus saimiri is a lymphotropic herpesvirus capable of immortalizing and transforming T cells both in vitro and in vivo. Immortalized and transformed T cells harbor several copies of the viral genome as a persisting genome. The mapping of the cis-acting genetic cis-acting segment (oriP) required for viral episomal maintenance is reported here. Viral DNA fragments that potentially contain oriP were cloned into a plasmid that contains the hygromycin resistance gene. After several round of subcloning followed by transfection, oriP was mapped to a 1.955-kb viral segment. This viral fragment permits stable plasmid replication without deletion or rearrangement as well as episomal maintenance without integration or recombination. The function of oriP depends on a trans-acting factor(s) encoded by the viral genome. The 1.955-kb viral segment includes a dyad symmetry region located between two small nuclear RNA genes and is located upstream of the dihydrofolate reductase gene homolog. Therefore, this oriP contains novel elements distinct from those of other DNA viruses.

A herpesvirus saimiri membrane protein required for interleukin-2 independence forms a stable complex with p56lck.

ORF-2, a 32-kDa viral protein expressed by herpesvirus saimiri-transformed lymphocytes, is essential for transformation and is expressed on the plasma membrane of transformed cells. The current work now shows that most (approximately 80%) of ORF-2 resides in the cytoplasm, while only a small portion protrudes from the cell surface. Expressed as a glutathione S-transferase fusion protein, ORF-2 was found to interact with a 56-kDa cellular protein in untransformed, herpesvirus saimiri-transformed, and Jurkat lymphocytes. Microsequencing proved that this protein is the lymphocyte-specific tyrosine protein kinase p56lck. Two regions of ORF-2 were found to be required for p56lck interaction. Current evidence suggests that the interaction of ORF-2 with p56lck plays a key role in the specific transformation of T lymphocytes to an interleukin-2-independent phenotype.

Immunoglobulin germline mu transcripts in acute myelogenous leukemia cells vary in splicing pattern and are heterogeneous.

Germline transcription of the immunoglobulin (Ig) locus is felt to play an important role in B cell differentiation. Similar transcripts are also found in neoplastic myeloid cells, but are of unknown significance. We have mapped these RNAs using reverse transcriptase polymerase chain reaction amplification. Unlike B cell transcripts, the majority of myeloid mu transcripts do not contain amplifiable enhancer sequence or complete 5' sequence. The extent of this deletion is related to the degree of myeloid maturation, with transcripts in the most primitive myeloid cells more closely resembling those in B cells. Variable 3'-splicing patterns are also observed in myeloid cells, unlike the single pattern identified in early B cells. Using primers which span the region from the 3' end of C mu 4 to the 5' end of the second membrane exon (M2), the splice sites between C mu 4 and M1, and between M1 and M2 have been analyzed. Our data suggest that factors important in initiation of germline mu transcription are present in both B lymphoid and myeloid cells, but that lineage-specific modifying factors alter this expression during myeloid maturation. Thus, the finding of Ig transcripts and other evidence of B lymphoid differentiation in acute myelogenous leukemia most likely reflects a retained common hematopoietic gene program.

A herpesvirus saimiri protein required for interleukin-2 independence is associated with membranes of transformed T cells.

A region of the herpesvirus saimiri genome encoding an mRNA with two open reading frames (ORFs) has been identified to be essential for transformation of T cells. Deletion of either ORF resulted in the loss of transforming ability. ORF-1 has been shown to code for a collagen-like oncoprotein. This study shows for the first time that the bicistronic mRNA can translate a 32-kDa protein from ORF-2. Polyclonal serum to ORF-2 was generated by using a glutathione fusion protein. Using this antiserum, ORF-2 was localized in cell membranes and is expressed on the outer cell membrane. The half-life of this membrane protein was found to be about 5.5 h. Limited sequence similarity was found between ORF-2 and interleukin-11; however, no secretion of ORF-2 protein was detected in supernatants from transformed cells. Further studies are required to investigate the potential interaction with the interleukin-11 receptor.

Expression of IL-2 and IL-4 in T lymphocytes transformed by herpesvirus saimiri.

Herpesvirus saimiri (H. saimiri) is a highly oncogenic lymphotropic herpesvirus which can immortalize T lymphocytes and cause tumors in rabbits and New World monkeys. T cells infected with strain 484-77 of group C express four viral U-like small RNAs (HSUR1-4) and a 1.2-kb mRNA which encodes open reading frames ORF-1 and ORF-2. ORF-1 encodes a collagen-like oncoprotein. Deletion mutation analysis showed that ORF-1 and ORF-2 are essential for IL-2 independent growth of human T cells infected with H. saimiri. An earlier study also demonstrated that H. saimiri-immortalized cells carry functional IL-2 receptors. The work presented in this report investigated whether IL-2 and IL-4 is produced by H. saimiri-immortalized T lymphocytes. Both IL-2 mRNA and IL-4 mRNA were detected in various monkey T cells as well as human peripheral blood lymphocytes infected with wild-type H. saimiri. Secretion of IL-2 was suggested by cyclosporin A inhibition. IL-4 secretion by monkey T cell cultures was demonstrated by a bioassay and inhibition of bioactivity by an antibody to IL-4. The data also show that recombinant IL-4 stimulate H. saimiri-immortalized T cells; thus, IL-4 receptors are expressed. However, antibodies to human IL-4, IL-4 receptor, or soluble IL-4 receptor did not curtail growth of transformed cells. T cells infected with ORF-1 and ORF-2 deletion mutants expressed no detectable IL-2 mRNA. ORF-1, ORF-2, HSUR1, and HSUR2, were all essential for expression of IL-4 mRNA. These data are consistent with the hypothesis that H. saimiri-immortalized monkey and human T lymphocytes proliferate through autocrine secretion of IL-2 and that ORF-1, ORF-2, and HSUR sequences of the virus are involved in expression of lymphokines.

A polycistronic transcript in transformed cells encodes the dihydrofolate reductase of herpesvirus saimiri.

Herpesvirus saimiri, an oncogenic gamma herpesvirus of primates, is the only eukaryotic virus that carries the entire metabolic gene set for a complex biochemical synthesis. Every element of the thymidine synthesis gene cascade is present in the virus, and their function is probably related to the uniquely high A + T content of the genome. Although one member of the gene set, dihydrofolate reductase (DHFR), is mapped in a region required for oncogenesis, very little is known of the expression and function of this gene in transformed cells. We report the expression of the DHFR sequence on a novel, unique tricistronic transcript in virally transformed tumor cells. The DHFR sequence is the first open reading frame on a 5.3 kb minor transcript. Alpha-amanitine sensitivity indicates that it is an RNA polymerase II transcript, and since it is also polyadenylated it appears to be a functional, relatively unstable (half-life 3 hr) mRNA. Initiation of transcription uniquely overlaps with the HSUR3 small RNA gene. Expression of the small transcript appears to be alpha-amanitine resistant, implicating polymerase III transcription. Together with the remarkably low-level expression of HSUR3 in tumor cells, the data may indicate transcription interference between two different RNA polymerases, with unusual overlapping regulation and initiation.

Induction of a herpesvirus saimiri small RNA AU binding factor (AUBF70) activity and lymphokine mRNAs by T cell mitogens.

Herpesvirus saimiri (H. saimiri) can transform T lymphocytes and cause lymphoid tumors in rabbits and New World monkeys. H. saimiri-immortalized T cells express IL-2 and IL-4. The putative oncogenes of a group C strain of H. saimiri have been mapped to a region of the unique L-DNA which includes genes encoding four U-like small nuclear RNAs (HSUR1-HSUR4). Jurkat T cells express a 70 kD RNA binding factor (AUBF70) which binds HSUR2. Here we examined AUBF70 expression in resting and mitogen-stimulated human peripheral blood T cells and its sequence specificity and subcellular distribution. Band-shift assays demonstrated that resting human T cells express low amounts of AUBF70 which is induced by mitogen treatment. IL-2 and IL-4 mRNAs were co-induced with AUBF70 suggesting that AUBF70 is a positive regulator of lymphokine gene expression. Normal resting, mitogen-stimulated, and leukemic Jurkat T cells all express AUBF70 with virtually identical V8 proteolytic enzyme digestion patterns. Northern blots demonstrated that HSUR1 and HSUR2 are localized both in the nucleus and cytoplasm. HSUR2 accumulate in the cytoplasm in the presence of actinomycin D, which is consistent with re-transport of HSURs to the nucleus by (an) unstable factor(s). We hypothesize that HSUR1 and 2 transport AUBF70 from the cytoplasm to the nucleus; in the nucleus, AUBF70 binds and stabilizes lymphokine transcripts. Increased stability of lymphokine mRNAs could contribute to oncogenic transformation induced by H. saimiri.

Herpesvirus saimiri small RNA and interleukin-4 mRNA AUUUA repeats compete for sequence-specific factors including a novel 70K protein.

A highly oncogenic strain of the lymphotropic tumour virus herpesvirus saimiri (HVS; strain 484-77) expresses four small RNAs (HSUR1 to 4) in high copy numbers in transformed T cells. In HSUR1 and HSUR2 the 5' terminal regions contain conserved AUUUA sequence repeats. The same AUUUA repeats occur in the 3' non-coding regions of growth factor, lymphokine and protooncogene mRNAs, and the sequence is involved in rapid mRNA degradation. We report here that by using a highly specific u.v. cross-linking method we identified a novel 70K binding factor with AUUUA sequence specificity. Non-radiolabelled competition and V8 protease analysis show that the protein can form a complex with the 3' non-coding region of interleukin-4 mRNA and bind the AUUUA repeats of a HVS small RNA. We also detected an AUUUA-specific minor 32K human protein with the same electrophoretic mobility as a marmoset factor implicated in growth factor mRNA destabilization. The findings are consistent with the hypothesis that the viral small RNAs can compete for factors involved in rapid degradation of growth factor mRNAs and may contribute to viral oncogenesis.

Small RNA expression from the oncogenic region of a highly oncogenic strain of herpesvirus saimiri.

Herpesvirus saimiri induces acute lymphomas and leukemias in primates and rabbits. Sequence divergence of the right end unique region of the genome classifies virus strains into three groups (A, B, and C), and previous studies have demonstrated correlation between DNA grouping and oncogenicity. In order to relate different oncogenicity to the underlying molecular mechanisms, we reported earlier the expression of a bicistronic mRNA from the oncogenic region in a highly oncogenic group C strain, and the present study is the first report on small RNA transcripts from the same region. The transcripts and 6.2 kbp on the oncogenic region were sequenced and characterized. We show that four U-type small RNAs are expressed in tumor cells transformed by this strain, in contrast to the seven small RNAs reported from a weakly oncogenic group A strain. Sequence comparisons between the two strains showed that the right end region of strain 484-77 of group C is about 1 kbp shorter. The conserved 5' AUUUA repeats of some small RNAs, and their proposed implication in lymphokine mRNA stabilization, are also discussed.

Expression of the collagen-like putative oncoprotein of Herpesvirus saimiri in transformed T cells.

Herpesvirus saimiri induces acute lymphomas and leukemias in New World primates and rabbits. Previous work revealed that a highly oncogenic group C strain 484-77 encodes and expresses a bicistronic mRNA in tumor-derived T cells and the first open reading frame (orf1) is highly homologous to collagen. With the aid of an antibody against a synthetic orf1 peptide, we now report that the orf1 collagen-like protein is expressed in rabbit tumor derived cell lines and in vitro transformed human and monkey T cells. The orf1 protein is expressed in vivo, as indicated by specific antibodies detected in the serum from a tumor-bearing rabbit.

IL-2 independent growth and cytotoxicity of herpesvirus saimiri-infected human CD8 cells and involvement of two open reading frame sequences of the virus.

Herpesvirus saimiri is a primate tumor virus and induces acute T cell lymphomas and leukemias in New World monkeys and rabbits. We show in this report that infection of human peripheral white blood cells with a group C strain 484-77 results in selective expansion of CD8 lymphocytes with strong cytotoxic activity and these cells do not require interleukin-2 (IL-2) for growth. Infected cell cultures, termed herpesvirus-activated killer (HAK) cells, have been continuously maintained for several months in tissue culture and these HAK cells contain multiple copies of stable circular viral episomes. The growth and cytotoxicity of HAK cells was found independent of IL-2. Analysis of deletion mutant infected cells suggests that at least two open reading frame sequences of a bicistronic mRNA encoded by the viral genome is involved in controlling IL-2 independence. This model could facilitate studies on growth regulation of human cytotoxic T cells that are important effector cells in immune responses against infectious diseases and cancer and should help us to elucidate the mechanism of transformation by H. saimiri oncogenes.