Temperature-sensitive transformation by an Abelson virus mutant encoding an altered SH2 domain

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the beta B5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34 degrees C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5 degrees C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.     

Human T-cell leukemia virus type 2 Tax protein induces interleukin 2-independent growth in a T-cell line

Background

Evolutionary analysis may serve as a useful approach to identify and characterize host defense and viral proteins involved in genetic conflicts. We analyzed patterns of coding sequence evolution of genes with known (TRIM5 α and APOBEC3G) or suspected (TRIM19/PML) roles in virus restriction, or in viral pathogenesis (PPIA, encoding Cyclophilin A), in the same set of human and non-human primate species.

Results and conclusion

This analysis revealed previously unidentified clusters of positively selected sites in APOBEC3G and TRIM5 α that may delineate new virus-interaction domains. In contrast, our evolutionary analyses suggest that PPIA is not under diversifying selection in primates, consistent with the interaction of Cyclophilin A being limited to the HIV-1M/SIVcpz lineage. The strong sequence conservation of the TRIM19/PML sequences among primates suggests that this gene does not play a role in antiretroviral defense.     

Phosphatidylinositol turnover and transformation of cells by Abelson murine leukaemia virus.

The transforming protein of the Abelson murine leukaemia virus encodes a protein-tyrosine kinase. Previously, we have shown that in Abelson-transformed cells, the Abelson kinase regulates the phosphoserine content of ribosomal protein S6. Phorbol 12-myristate 13-acetate (TPA), which activates protein kinase C, induces the phosphorylation of S6 at the same five phosphopeptides as found in S6 isolated from Abelson-transformed cells. We have investigated three models whereby the Abelson kinase might regulate S6 phosphorylation via the activation of protein kinase C. First, the Abelson kinase could phosphorylate protein kinase C on tyrosine. However, we do not detect significant amounts of phosphotyrosine in protein kinase C in vivo. Second, it has been suggested that protein-tyrosine kinases might phosphorylate phosphatidylinositol. This could increase the intracellular levels of diacylglycerol and thereby activate protein kinase C. Our data strongly suggest that direct phosphorylation of phosphatidylinositol by the Abelson protein-tyrosine kinase has no physiological role. Third, an indirect activation of protein kinase C may occur via an increase in the rate of phosphoinositide breakdown. We have found that phosphoinositide breakdown appears to be constitutively activated in Abelson-transformed cells. The implications of these observations are discussed with regard to S6 phosphorylation and the mechanism of Abelson-induced transformation.     

Growth of an interleukin 2/interleukin 4-dependent T cell line induced by granulocyte-macrophage colony-stimulating factor (GM-CSF)

Lymphokine activities in conditioned medium from activated helper T cell lines are most commonly defined by the proliferation of "specific" lymphokine-dependent cell lines. Various sublines of IL 2-dependent (and ostensibly specific) HT-2 and CTLL cells have now been shown to proliferate in response to BSF-1/IL 4 as well. After activation with antigen or mitogen, D10.G4.1, an antigen-specific cloned T helper cell that has recently been shown to produce IL 4 but not IL 2, secretes two distinct cytokines that induce the growth of HT-2 cells. These "T cell growth factors" (TCGF) can be separated by reversed phase high-performance liquid chromatography (RP-HPLC). The TCGF activity of one of these factors can be blocked by 11B11, an antibody specific for IL 4. The second TCGF activity is not affected by 11B11 or by antibodies specific for IL 2. This TCGF activity can be neutralized by a goat polyclonal antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF), and has a RP-HPLC elution profile identical to that of recombinant GM-CSF. Recombinant GM-CSF induces both proliferation and long-term growth of HT-2 but not CTLL cells, and this activity can be neutralized by the same antibody to GM-CSF. GM-CSF is best known as a factor that induces the maturation and growth of granulocytes and macrophages from bone marrow-derived hematopoietic precursor cells. The ability of GM-CSF to induce the growth of certain T cell lines indicates that this molecule may play a role in T cell-mediated immune responses, either as an autocrine growth factor or a paracrine stimulus from both lymphoid and nonlymphoid tissues that produce this cytokine.     

Establishment of a cyclic adenosine monophosphate-dependent growing human T-cell line derived from an interleukin-2-dependent cell line

An adenosine 3',5'-cyclic monophosphate (cAMP)-dependent growing cell line called CT-Mat was established by the long-term cultivation of an interleukin-2 (IL-2)-dependent human T-cell line, ILT-Mat, in the presence of cholera toxin instead of IL-2. CT-Mat cells can grow in the medium containing either cholera toxin or forskolin or cAMP derivatives. Although the CT-Mat cell line can still grow dependent on IL-2, the forskolin-induced growth of CT-Mat cells was demonstrated not to be mediated by an autocrine mechanism of IL-2 or any other growth factor. The intracellular cAMP level was elevated by treatment with the chemical agents but little by treatment with IL-2. These suggest that cAMP transduces intracellular growth signals different from those through the IL-2 receptor in an IL-2-dependent T-cell line CT-Mat.     

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.     

Abelson virus abrogation of interleukin-3 dependence in a lymphoid cell line.

Among several tyrosine-protein kinases, only v-abl could abrogate interleukin 3 dependence of a lymphoblastoid cell line; v-src and v-fps proteins gave partial or no interleukin 3 independence, respectively. Lymphokine independence was achieved via a nonautocrine mechanism. Direct involvement of c-myc in this process was not evident.     

Characterization of interleukin 2-dependent cytotoxic T-cell clones. IV. Production of alpha, beta and gamma interferons and interleukin 2 by Lyt-2+ T cells

The production of alpha, beta and gamma interferons (IFN) and interleukin 2 (IL-2) by Lyt-2+-dependent cytotoxic T-cell lines/clones was investigated. Cloned and uncloned T-cell lines specific for H-2Dd or the unique RL male 1 leukemia antigen were studied. After infection with Sendai virus (SV) or Newcastle disease virus (NDV) all cell lines produced IFN-alpha and -beta. Induction of IFN-gamma was attempted with the mitogens Con A, PHA, PWM, SEA, and SEB, with poly(I:C), with antibodies Lyt-1.2, -2.2, and Thy-1.2, or with the target cells Meth A (H-2Dd+) and RL male 1. All mitogens were effective inducers. However, the antibodies and poly(I:C) were not. One uncloned RL male 1-specific cell line CTLL-RP, produced IFN-gamma after induction with RL male 1. Production of IFN-alpha, beta depended on IL-2, whereas production of IFN-gamma did not, although addition of highly purified IL-2 increased IFN-gamma production even in the absence of other inducers. Crude IL-2 inhibited the production of IFN-gamma but not IFN-alpha, beta. In response to mitogens, some T-cell clones also produced IL-2. The results demonstrate that Lyt-2+ cells can produce a broad spectrum of lymphokine activities after appropriate stimulation. Their availability now affords us the opportunity to study the regulation of lymphokine production at the clonal level.     

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.     

Interleukin 2 rapidly stimulates synthesis and breakdown of polyphosphoinositides in interleukin 2-dependent, murine T-cell lines

Several T-cell functions are controlled by the regulatory peptide interleukin 2 (IL-2). Binding of IL-2 with specific receptors has been well documented, but the molecular mechanism by which IL-2/IL-2 receptor interaction is transduced is not known. We have found that treatment of IL-2-dependent T-cell lines with IL-2 is followed by a rapid stimulation of inositol phospholipid metabolism, as determined by isotopic methodology employing myo-[1,2-3H]inositol. Increased incorporation of the metabolic precursor into phosphatidylinositol and phosphatidylinositol 4-monophosphate, together with the appearance of radiolabeled phosphatidylinositol 4,5-bisphosphate, occurred within minutes of treatment with IL-2 of factor-dependent CT6 cells. Analysis of labeled water-soluble compounds from prelabeled cells indicated a rapid (within 1 min) stimulation of inositol phospholipid hydrolysis following IL-2 treatment. Increased recovery of [3H] inositol phosphates and appearance of [3H]inositol trisphosphate were observed after treatment with IL-2 of CT6 cells, as well as of a second IL-2-dependent cell line, CTB6. These findings suggests that inositol phospholipid-derived metabolites (i.e. diacylglycerol and inositol trisphosphate) may be part of the mechanism by which certain IL-2 signals are transduced.     

Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation.

Abelson murine leukemia virus transforms both lymphoid cells and fibroblasts in vitro and induces a unique type of thymus-dependent lymphoma in vivo. Four fibroblast-transforming strains of Abelson murine leukemia virus were identified, based on the sizes of the Abelson murine leukemia virus-specific phosphoproteins produced by these isolates. Two of these strains, the standard P120- and the P160-producing viruses, transformed lymphoid cells efficiently in vitro and induced Abelson disease in vivo. Two other strains, which synthesized small Abelson murine leukemia virus-specific proteins with molecular weights of 90,000 (P90) and 100,000 (P100), transformed lymphoid cells very poorly both in vitro and in vivo. The reduced oncogenic potentials of these isolates were correlated with a high level of synthesis of fairly unstable P90 and P100. In addition, neither P90 nor P100 functional efficiently in protein kinase assays. The correlation of abnormal metabolism and deficient protein kinase activity with the reduced oncogenic potentials of these virus strains supported a direct role for these proteins and the kinase activity in transformation. Furthermore, these results suggested that the requirements for lymphoid cell transformation and fibroblast transformation are different.     

Regulation of growth of an interleukin 2 (IL-2)-dependent murine T-cell clone (HT-2) in a defined serum-free medium

In this study, we demonstrate that an IL-2-dependent T-cell clone (HT-2) can be grown in a serum-free medium (HB101) with defined additives at rates comparable to those which can be obtained in serum-containing medium. Further, we show that cells cultured in the serum-free medium in the absence of IL-2 arrest growth in the G1 portion of the cell cycle, and that these arrested cells can be stimulated to reenter the cell cycle upon the addition of IL-2 to the culture medium. Growth of these cells in the absence of serum requires the presence of IL-2 as well as other hormones and growth factors and 2-mercaptoethanol. HT-2 cells have been grown continuously in the serum-free medium for periods of up to 1 month.     

T-cell transformation by Marek's disease virus

Marek's disease virus (MDV) is an avian herpesvirus that causes rapid development of T-cell lymphomas in chickens. The MDV genes currently thought to be involved in lymphomagenesis include a bZIP transactivator that is homologous to fos and jun oncogenes but do not appear to have counterparts in other oncogenic herpesviruses.     

Expression of interleukin 2 receptors on interleukin 3-dependent cell lines

Several mouse IL 3-dependent cell lines, IC2, LT4, FDC-P2, and PB-3C, derived from spleen or bone marrow cells were shown to express low affinity receptors for IL 2 (Kd; 0.5 to 8 X 10(-8) M). High affinity receptors for IL 2 were not detected on the IL 3-dependent cells within the experimental limitation of this study. The clones did not respond to IL 2 at all at the concentration as high as 25 micrograms/ml. The number of the receptors expressed on those clones was estimated to be 0.2 to 2 X 10(5)/cell, which is comparable with the number of those on IL 2-dependent T cell clones. Expression of IL 2 receptor was confirmed in mRNA levels for both IC2 and LT4 cells. A relatively low level expression of one (4.5 Kb) of four IL 2 receptor mRNA species was observed with those IL 3-dependent clones compared with IL 2-dependent T cells. It seems that these low affinity receptors may be expressed on IL 3-dependent cells that undergo differentiation or maturation in mast cell and some myeloid cell lineages.     

Voltage-gated K+ channels in the mouse interleukin 3-dependent cell line,FDC-P2

Summary The electrical properties of a mouse interleukin (IL)-3-dependent cell line, FDC-P2, were examined using the tightseal whole-cell clamp technique. Under current clamp conditions with 140mM K⁺ in the pipette, the cells had a resting potential of –30 mV. Under voltage-clamp conditions, a transient outward current was elicited upon depolarization from a holding potential of –80 mV. The current was activated at potentials more positive than –10 mV and had a delayed-rectifying property. It showed rapid activation and slow inactivation during command steps. The current was abolished by Cs⁺ in the pipette, indicating that K⁺ is the charge carrier. The K⁺ current was suppressed by tetraethylammonium withK _i of <0.1mM and was not affected by scorpion toxin. Recovery from inactivation was steeply voltage dependent: As the holding potential was more hyperpolarized, the recovery became faster. Thus, with a holding potential of –80 mV, the current showed slight use-dependent inactivation, while the current decreased prominently by repetitive depolarization at a holding potential of –40 mV. These properties of the K⁺ current are similar to those of thel-type K⁺ channel current in mature T lymphocytes. The K⁺ current in FDC-P2 cells was dramatically reduced after culture in the IL-3-free medium for 1–2 days. When IL-3 was re-added to the medium, the current was re-expressed. These observations suggest that expression of the K⁺ current depends on extracellular IL-3, and that the current may play some roles in proliferation of these cells.     

Modulation of interleukin 2 internalization and interleukin 2-dependent cell growth by antireceptor antibodies

The growth factor interleukin 2 (IL2) binds to and is internalized together with high-affinity surface receptors present on lymphoid cells. This endocytosis thus results in down-regulation of the receptors. However, it is not known if the internalization is relevant to the induction of cell growth. In the present study a rat monoclonal antibody to the P55 chain of the IL2 receptor was used to examine the role of receptor internalization in the IL2-dependent autocrine human tumor T cell line IARC 301. When given alone, this antibody did not inhibit IL2 binding, internalization, or IL2-dependent cell proliferation. However, crosslinking by anti-rat immunoglobulins, which did not affect binding of the growth factor, inhibited both IL2 internalization and cell proliferation. Besides offering a novel means for the specific inhibition of the uptake of IL2 bound to IL2 high-affinity receptors, the results are compatible with the association of this receptor-ligand uptake to the growth stimulation by IL2.     

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.     

Site-directed deletions of Abelson murine leukemia virus define 3'' sequences essential for transformation and lethality.

Abelson murine leukemia virus (A-MuLV) encodes a single protein with tyrosine kinase activity that can transform fibroblast cell lines in vitro and lymphoid target cells in vitro and in vivo. Expression of kinase-active A-MuLV protein can result in a deleterious effect on transformed fibroblast populations, leading to cell death or selection for nonlethal mutants of the virus. These mutants retain expression of the kinase activity but have lost large portions of the carboxy terminus of the Abelson protein. To more precisely map the sequences involved in this lethal effect, we have isolated a series of site-directed deletions from a DNA clone of the P160 wild-type strain of A-MuLV. In addition, a number of unexpected, spontaneous deletions occurring during transfection of NIH 3T3 cells were isolated. These deletions result in expression of carboxy-terminal truncated forms of the A-MuLV protein ranging from 130,000 to 84,000 in molecular weight. Analysis of the transforming and lethal activities of each mutant recovered in its RNA viral form shows that the transformation-essential and lethal-essential sequences do not overlap. These data and our previous work suggest that a function carried by the carboxy-terminal region of the A-MuLV protein acts in cis with the kinase-essential region to mediate the lethal effect.