Virus-induced Transformation Research Articles

Influence of the Epstein-Barr virus nuclear antigen EBNA 2 on the growth phenotype of virus-transformed B cells.

Epstein-Barr virus (EBV) isolates show sequence divergence in the BamHI YH region of the genome which encodes the nuclear antigen EBNA 2, a protein thought to be involved in the initiation of virus-induced B-cell transformation; type A isolates (such as B95-8 EBV) encode a 82- to 87-kilodalton EBNA 2A protein, whereas type B isolates (such as AG876 EBV) encode an antigenically distinct 75-kilodalton EBNA 2B protein. In the present work 12 type A isolates and 8 type B isolates have been compared for their ability to transform resting human B cells in vitro into permanent lymphoblastoid cell lines. Although the kinetics of initial focus formation was not markedly dependent upon the EBNA 2 type of the transforming virus, on subsequent passage type A virus-transformed cells (type A transformants) yielded cell lines much more readily than did type B transformants. Direct comparison between the two types of transformant revealed clear differences in several aspects of growth phenotype. Compared with type A transformants, cell lines established with type B virus isolates consistently displayed an unusual growth pattern with poor survival of individual cells shed from lymphoblastoid clumps, a lower growth rate and a greater sensitivity to seeding at limiting dilutions, and a significantly lower saturation density that could not be corrected by supplementation of the medium with culture supernatant containing B-cell growth factors. This is the first direct evidence that, in EBV-transformed B-cell lines, the EBNA 2 protein plays a continuing role in determining the cellular growth phenotype.

Epstein-Barr virus-induced transformation and immune surveillance

The inhibitory effects of pure recombinant human interferons αA and γ (reIFN-αA and -γ) on Epstein-Barr virus (EBV) infection of a human EBV-negative B cell line, BJAB, and of normal adult B lymphocytes were studied. With pretreatment for 24 h, both types of reIFNs were effective in suppressing the production of EBV specific nuclear antigen (EBNA-1) in BJAB cells 24 h after EBV-infection, as determined by the immunoblotting technique. ReIFN-αA was, however, a much more potent inhibitor than reIFN-γ. With treatment starting 1 h after EBV infection, both types of reIFNs were less effective in the suppression of EBNA production. Neither of the reIFNs showed any inhibitory effect on EBNA production in the latently EBV-infected cell lines, Raji and Daudi. These results suggest that reIFNs act in the early phase of EBV infection. Both types of reIFNs were also effective in inhibiting EBV infection of normal adult B lymphocytes as demonstrated by a reduction both in [3H]thymidine incorporation 6 days after EBV infection and in the total number of proliferating cells 21 days after EBV infection. Again, reIFN-αA showed a greater inhibitory effect than reIFN-γ. We also showed that in BJAB cells, reIFN-αA strongly induced (2'–5')oligoadenylate synthetase activity, whereas reIFN-γ increased the surface expression of HLA class I antigens.

1] Purification of human platelet-derived growth factor

Publisher Summary This chapter focuses on purification of human platelet-derived growth factor (PDGF). PDGF is probably a heterodimer of one A chain and one B chain, although the possibility that homodimers occur has not been ruled out. Determination of a partial amino acid sequence of PDGF revealed that the B chain is almost identical to part of p28 sis the transforming protein of simian sarcoma virus. This indicates that a PDGF-like growth factor is involved in simian sarcoma virus-induced cell transformation. The A chain of PDGF showed approximately 60% homology to the B chain indicating that the gene sequences encoding the two PDGF chains have evolved from a common ancestral gene. The procedure to purify PDGF from human platelets described in the chapter is based on accumulated experience from PDGF purification in laboratory and has been used in its present form for the last 6 years.

Effects of S-adenosylhomocysteine and analogs on Epstein-Barr virus-induced transformation, expression of the Epstein-Barr virus capsid antigen, and methylation of Epstein-Barr virus DNA.

S-Adenosylhomocysteine was found to have no effect on Epstein-Barr virus-induced transformation of B-lymphocytes and to stimulate viral capsid antigen expression only slightly in the FF41-1 cell line. In contrast, the S-adenosylhomocysteine analogs sinefungin and S-isobutyladenosine inhibited Epstein-Barr virus transformation and induced a significant increase in the numbers of cells expressing the viral capsid antigen. An inverse relationship between levels of viral DNA methylation and gene expression was demonstrated.

T cell receptor-homologous mRNA from a suppressor T cell clone directs the synthesis of antigen-specific suppressive products.

The LH8-105 T cell clone, obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific mouse suppressor T lymphocytes, constitutively releases in the culture supernatant products able to induce specific suppression of the immune response to HEL. LH8-105 cells rearrange and express the genes encoding the alpha and beta chains of the antigen-specific T cell receptor and display membrane T cell receptor structures. LH8-105 mRNA specific for the alpha and beta chains of the T cell receptor were positively selected on filter-bound cDNA encoding constant regions of alpha and beta chains, eluted and translated into Xenopus laevis oocytes. Translation products were then tested in vivo for specific suppression of the anti-HEL antibody response. LH8-105 culture supernatant and translation products of LH8-105 poly(A)+ RNA or a mixture of alpha and beta chain-homologous mRNA induce HEL-specific suppression whereas translation products of LH8-105 mRNA eluted from irrelevant DNA or unrecombined mRNA eluted from alpha and beta cDNA do not exert suppressive activity. These results indicate that alpha and beta chain-homologous mRNA expressed in LH8-105 cells direct the synthesis of molecules able to induce HEL-specific suppression.

Human B lymphocytes immortalization by Epstein-Barr virus in the presence of cyclosporin A.

A simple method was devised for the establishment of continuous lymphoblastoid cell lines (LCL). Unfractionated mononuclear cells collected from healthy donors were infected in vitro by Epstein-Barr Virus (EBV) (strain B95-8) under specific conditions: an immunosuppressive drug, Cyclosporin-A (CS-A, Sandoz), associated with the use of a feeder layer (MRC-5) led to 100% efficiency of LCL establishment. A bank of 400 LCL was set up for completion of genetic studies. Regression and kinetics of virus-induced transformation were monitored and related to donors' EBV immune status. Mean time of LCL establishment and probability of regression among seropositive donors were not linked to any given value titer of antibodies against Viral Capsid Antigen (VCA) or against Epstein-Barr Nuclear Antigen (EBNA). However, when the anti-VCA:anti-EBNA ratio was considered, this parameter seemed to be linked to the kinetics of transformation but not to the probability of regression. Once LCL are established, large quantities of human cells can be produced. The complete cellular DNA is available so that any part of it can be scrutinized. Moreover, some of the phenotypic characteristics of these B cells can be used for a wide range of investigations.

Does virus-induced transformation affect the activity of amino acid transport system L ?

Does virus-induced transformation affect the activity of amino acid transport system L ?

Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation.

The transforming activity of polyoma virus middle-T antigen is believed to be dependent on its ability to form a complex with the cellular tyrosine protein kinase, pp60c-src. This hypothesis is based on observations of mutants of middle-T which demonstrated a correlation between these two activities. To investigate further the significance of pp60c-src association in transformation by middle-T, a series of deletion and point mutants were constructed around the NG59 lesion since this region has been implicated in pp60c-src binding. Analysis of the middle-T variants revealed a complete correlation between the presence of associated activated pp60c-src and the ability to transform. Further, this ability of pp60c-src to associate with middle-T may depend on the presence of a beta-turn between amino acids 177 and 180. The results indicate the NG59 phenotype results from the introduction of an isoleucine residue between amino acids 177 and 178 rather than the transition mutation at 179. The mutant MG1 is a single point mutation (at residue 180) and represents the smallest change in the middle-T which abolishes both the transformating and kinase activity of middle-T. Taken together, the data suggest the region surrounding the NG59 lesion is involved in the formation of an active complex between middle-T and pp60c-src and strongly suggest that this association is an absolute requirement for polyoma virus-induced transformation.

The ras oncogene product p21 is not a regulatory component of adenylate cyclase

Harvey (Ha-MSV) and Kirsten (Ki-MSV) murine sarcoma viruses induce tumours in animals and transform various cells in culture because of the expression of the ras oncogene product, p21 (ref. 1). Proto-oncogenes homologous with these genes are highly conserved evolutionarily and activated ras oncogenes have been detected in many human cancers. Whether c-ras oncogenes are directly responsible for human carcinogenesis is uncertain; however, it is clear that p21 mediates virus-induced transformation, although by an unknown mechanism. Epithelial and fibroblast cell lines transformed with Ha-MSV and Ki-MSV express p21 (ref. 8) and exhibit reduced adenylate cyclase activity. Like the guanine nucleotide regulatory proteins, Ns and Ni, which mediate stimulation and inhibition, respectively, of adenylate cyclase, p21 is a membrane-associated GTP binding protein, which exhibits GTPase activity. These similarities suggest that p21 and the adenylate cyclase regulatory proteins are related in cellular function, and that p21 depresses adenylate cyclase by inhibiting the activity of Ns or acting as Ni. We have therefore now examined the structural and functional similarities between p21 and Ns and Ni and find no evidence that p21 regulates adenylate cyclase activity by acting as one of these regulatory proteins.

Cellular site and mode of Fv-2 gene action.

The Fv-2 genotype of erythroid progenitors directly determines whether they will undergo viral-induced transformation. This conclusion was reached from studies of allophenic mice compounded from congenic C57BL/6 strains differing at Fv-2 and an enzyme marker (GPI). Infection of these Fv-2ss in equilibrium Fv-2rr mosaic animals with the polycythemic strain of Friend virus results in the development of Friend disease. Concomitant with disease symptoms is a shift in the mosaic composition of the erythrocytes in favor of those of the susceptible strain. The observed viral-induced shift in the erythrocyte composition is paralleled by a similar change in the mosaic composition of the CFU-E pool but not the primitive (d8) BFU-E pool. Thus, with regard to this particular Fv-2 phenotype (susceptibility to FV-P-induced cellular hyperplasia), Fv-2 manifests itself specifically in the erythroid lineage, either in mature (d3) BFU-E or CFU-E.

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.

Disulfide-linked surface molecules of monoclonal antigen-specific suppressor T cells: evidence for T cell receptor structures.

By two-dimensional polyacrylamide gel electrophoresis analysis under nonreducing/reducing conditions, five proteins with interchain disulfide bridges are revealed on the surface of the suppressor T cell lymphoma line LH8-105 obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme-specific suppressor T lymphocytes. Two disulfide-linked surface proteins expressed by LH8-105 cells have been positively identified by immunoprecipitation with specific antisera. The major labeled membrane protein of LH8-105 cells is the murine leukemia virus env glycoprotein gp70. The second disulfide-linked molecule identified on LH8-105 cells has a molecular mass of 84 kDa under nonreducing conditions and 42 kDa after reduction, and is immunoprecipitated by an antiserum which recognizes the T cell receptor for antigen. A disulfide-linked molecule of a similar molecular mass is also immunoprecipitated from surface-labeled LH8-105 cells by a rabbit antiserum directed against a synthetic peptide predicted from the nucleotide sequence of a cDNA clone encoding the beta chain constant region of a helper T cell hybridoma. Therefore, a dimeric structure comparable to the T cell receptor expressed by cytotoxic and helper T cells is present on the cell surface of these monoclonal antigen-specific suppressor T cells.

Retrovirus-induced expression of interleukin 2 receptors on cells of human B-cell lineage.

We provide direct evidence for interleukin 2 receptor (IL2R) induction by human T-cell leukemia/lymphoma virus (HTLV) in human B-cell lines. A lymphoblastoid B-cell line (LCL-Ter) was established by Epstein-Barr virus-induced transformation of peripheral blood lymphocytes derived from a healthy HTLV carrier and cloned in vitro. HTLV gp21 and/or p19 antigens were detected in eight LCL-Ter clones, all of which also expressed the IL2R antigens Tac and Hiei (defined by monoclonal antibodies). However, five other LCL-Ter clones, which were negative for the HTLV antigens, did not express the IL2R antigens. Furthermore, when the IL2R-negative B-cell line LCL-Kan, derived form a normal donor, was cocultured with HTLV-producer cells, three HTLV-carrying clones were obtained and found to constitutively express IL2R. IL2R induced by HTLV on these B-cell lines bound recombinant interleukin 2 and were similar in apparent molecular mass (approximately equal to 60 kDa) to the IL2R of peripheral blood lymphocytes stimulated with phytohemagglutinin.

Microplate method for retrovirus-induced transformation of normal human T-cells

An adult T-cell leukemia virus (ATLV) producer cell line (TL-Su) derived from a healthy ATLV-carrier, was used for virus-induced transformation of normal human T-cells. The transformation was carried out on microplates by co-cultivation of a limiting number of normal peripheral blood leukocytes (PBL) as transformed target cells and 2 × 10 4/well of TL-Su cells irradiated lethally. The frequency of establishment of transformed cell lines correlated with the dose of PBL. The transforming target cell number was estimated to be approximately one per 500 PBL by the microplate method using TL-Su effector cells. When 1 × 10 3 PBL per well were plated, more than 90% of wells showed immortalizing transformation of PBL. Twenty transformed cell lines obtained from microplates with 500 PBL per well were shown to be of T-cell lineage. Ten of them were defined as belonging to the helper/inducer T-cell subset with Leu 3a antigen, and 4 were demonstrated to be of the cytotoxic/suppressor T-cell subset with Leu 2a antigen. The transformed cells always expressed Tac and OKT9 antigens (considered to be IL2 and transferrin receptors respectively). ATLV antigens were also expressed in all these cell lines tested, but their molecular species detected by radioimmunoprecipitation varied regardless of PBL donors and T-cell subsets. We propose this microplate transformation method as an advantageous system for obtaining abundant T-cell lines and for screening their immunological functions.

T-cell-mediated regression of “spontaneous” and of Epstein-Barr virus-induced B-cell transformation in vitro: Studies with cyclosporin A

The regression of Epstein-Barr (EB) virus-transformed B-cell outgrowth which is seen in experimentally-infected cultures of blood mononuclear (UM) cells from healthy seropositive donors can be abolished in medium containing the T-cell-suppressive agent cyclosporin A (CSA) at concentrations of 0.05 μg/ml and above. CSA mediates its effect within the first 4 days post-infection of the UM cells and this prevents subsequent in vitro generation of the EB virus-specific cytotoxic-T-cell response which normally brings about regression. Regression can be fully restored by supplementing the CSA-treated culture with interleukin 2 (IL-2)-containing culture supernatants or indeed with purified IL-2 itself, suggesting that CSA mediates its effect in this system through inhibiting the endogenous production of IL-2 which is required to amplify the virus-specific cytotoxic response. “Spontaneous transformation” to EB virus genome-positive lymphoblastoid cell lines in noninfected cultures of UM cells from healthy seropositive donors, though rare in normal medium, is enhanced to such a degree in the presence of CSA that, for many donors, the phenomenon becomes titratable against input cell dose across the 2.0 × 10 6−2.5 × 10 5 cells/culture range. Cell mixing experiments suggest that the spontaneously transformed cell lines which arise with such efficiency under these conditions do so not by direct in vitro outgrowth of progenitor cells transformed by the virus in vivo, but by a two-step mechanism involving virus release and secondary infection in vitro.

Stages in development of mink cell focus-inducing (MCF) virus-accelerated leukemia in AKR mice.

Flow cytometric techniques involving correlated dual parameter analysis of fluorescence and light scatter and transplantation bioassays were used to describe a series of cellular changes in thymus of young (1-4 mo old) AKR mice during development of mink cell focus-inducing (MCF) virus-accelerated leukemia. Three stages of leukemogenesis were defined before appearance of frankly leukemic mice. Stage 1, apparent 28-40 d after injection of MCF 69L1 virus, represented steady-state infection of thymocytes by MCF virus without apparent change in light scatter properties of the cells or in expression of alloantigens Thy-1, Lyt-1, Lyt-2, L3T4a, B2A2, or H-2K on the major thymocyte subpopulations. Expression of MCF virus was highest in the population of small cortical thymocytes. Stage II was observed at highest frequency 50-60 d postinjection and represented the emergence of a clonal population of cells with transformed properties which could be resolved from normal thymocytes by light scatter and expression of B2A2, H-2K, and gp70 antigens. Stage III was observed at highest frequency at 70 d postinjection, when considerable enlargement of thymus had occurred, and appeared to represent the outgrowth of fully transformed cells that replaced the normal thymocyte subpopulations. The alloantigen phenotype of blast cells from frankly leukemic mice did not differ qualitatively from that of stage II or stage III cells but displayed considerable heterogeneity with respect to quantitative expression of alloantigens and gp70. At least two populations of leukemic blasts could be resolved in the majority of primary thymomas analyzed. It is unclear whether these populations represent the outgrowth of independent clones of transformed cells or if they are related in some way. Our data are consistent with MCF virus-induced transformation of cells in the lineage to small peanut agglutinin-positive, cortisone-sensitive thymocytes, a subpopulation that predominates in the thymus and which is thought to be destined for cell death in situ.

Immunoregulation of lysozyme-specific suppression. II. Hen egg-white lysozyme-specific monoclonal suppressor T cell factor suppresses the afferent phase of delayed-type hypersensitivity and induces second-order suppressor T cells.

Culture supernatant from a monoclonal T cell lymphoma line (LH8-105) obtained by radiation leukemia virus-induced transformation of hen egg-white lysozyme (HEL)-specific suppressor T lymphocytes is able, when injected into mice, to specifically suppress the delayed-type hypersensitivity (DTH) reaction induced by HEL. The suppressor T cell factor (TsF) exhibits fine antigenic specificity since it suppresses the DTH response induced by HEL without affecting the DTH response induced by ring-necked pheasant egg-white lysozyme (REL), a lysozyme closely related to HEL. Conversely, LH8-105 TsF is able to suppress the DTH response induced by human lysozyme, distantly related to HEL but sharing a common epitope critical for induction of suppressive activity. The fine antigenic specificity of LH8-105 TsF for a restricted epitope on the HEL molecule is confirmed by binding to HEL but not to REL immunosorbents. This TsF also bears I-J determinants, as demonstrated by binding to monoclonal anti-I-J immunosorbents, and it suppresses the afferent but not the efferent phase of the DTH response to HEL. The afferent suppression is controlled by genes apparently mapping in the I-J subregion of the H-2 complex since I-J-incompatible mice are not suppressed by LH8-105 TsF injection. This inducer-type TsF induces second-order effector suppressor T cells only in HEL-primed mice indicating the primary role of antigen, in association with H-2 (I-J) products, in the afferent portion of this suppressive circuit.

Avidin is induced in chicken embryo fibroblasts by viral transformation and cell damage.

Synthesis and secretion of avidin was studied in cultured chicken embryo fibroblasts infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature-sensitive for transformation, OK-10 virus) or a nontransforming retrovirus (RAV-1). Avidin was detectable in both transformed and untransformed cultures, and was identical to chicken egg white avidin by several criteria: biotin-binding, heat-induced biotin exchange, subunit size (mol. wt. 15 600), immunoprecipitation of metabolically labeled proteins and immunoblotting. Transformation increased the production of avidin up to 50-fold, but several experiments suggested that the induction was not a direct consequence of virus-induced cell transformation. The production of avidin seemed to relate to cellular damage both in cultures of virus-transformed and of normal fibroblasts. It may represent a response to cellular damage and viral transformation may activate the process.

Production of EB virus by normal human nasopharyngeal epithelial cells exposed to the virus in vitro

Summary The infectability of normal human nasopharyngeal epithelial cells by EB virus has been investigated using a new culture method in which the cells grow in proliferating sheets shedding large, flat, differentiated cells into the medium. Cultures from five different biopsy samples were exposed to the virus by rocking in a virus preparation on one occasion, or by adding virus to the medium daily for several weeks, or by co-cultivation with lethally X-irradiated virus-producing lymphoid cells. There was no evidence of virus-induced transformation, expression of EB virus nuclear antigen or production of EB-virus capsid antigen (VCA), in any uninoculated control or test cell sheets. However, a small number of cells shed from the sheets from one biopsy showed definite VCA staining after co-cultivation with virus-producing cells, and electron microscopy of parallel samples revealed cytoplasmic herpesvirus nucleocapsids beside bundles of keratin. The significance of this EB-virus production by such squamous epithelial cells is discussed.

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.