Reticuloendotheliosis Virus Strain T Research Articles

Reticuloendotheliosis Virus Strain T Induces miR-155, Which Targets JARID2 and Promotes Cell Survival

The oncogenic microRNA miR-155 is upregulated by several oncogenic viruses. The precursor of miR-155, termed bic, was first observed to cooperate with myc in chicken B-cell lymphomas induced by avian leukosis proviral integrations. We identified another oncogenic retrovirus, reticuloendotheliosis virus strain T (REV-T), that upregulates miR-155 in chicken embryo fibroblasts. We also observed very high levels of miR-155 in REV-T-induced B-cell lymphomas. To study the role of miR-155 in these tumors, we identified JARID2/Jumonji, a cell cycle regulator and part of a histone methyltransferase complex, as a target of miR-155. The overexpression of miR-155 decreased levels of endogenous JARID2 mRNA. We confirmed that miR-155 directly targets both human and chicken JARID2 by assaying the repression of reporters containing the JARID2 3'-untranslated regions. Further, the overexpression of a sponge complementary to miR-155 in a tumor cell line increased endogenous JARID2 mRNA levels. The overexpression of JARID2 in chicken fibroblasts led to decreased cell numbers and an increase in apoptotic cells. The overexpression of miR-155 rescued cells undergoing cytopathic effect caused by infection with subgroup B avian retroviruses. Therefore, we propose that miR-155 has a prosurvival function that is mediated through the downregulation of targets including JARID2.

Resistance to Salmonella enteritidis Organ Invasion in Day-Old Turkeys and Chickens by Transformed T-Cell Line-Produced Lymphokines

We previously reported an increased resistance to Salmonella enteritidis (SE) organ invasion in chicks and turkey poults injected prophylactically with SE-immune lymphokines (ILK). In the present study, concanavalin A (Con-A)-activated splenic T cells isolated from SE-hyperimmunized hens were transformed in vitro with reticuloendotheliosis virus strain T (REV-T) (chicken syncitial virus). These transformed T cells were then maintained as a long-term (> 1 yr) cell line for the harvest of immune lymphokines (VILK). The efficacy of VILK to protect turkey poults and chicks against SE organ invasion and the correlation between organ invasion and peripheral blood heterophilia were then evaluated. Three groups of day-old poults and chicks were injected intraperitoneally with either phosphate-buffered saline (PBS; group A), ILK (group B), or VILK (group C). Thirty minutes postinjection, poults and chicks were challenged per os with 5 x 10(5) colony-forming units (CFU) SE and 5 x 10(4) CFU SE, respectively. At 24 hr posttreatment, birds in groups A, B, and C were euthanatized and liver samples were cultured for the presence of SE. Both the VILK- and ILK-treated turkeys and chicks had significant reductions in organ invasion when compared with the PBS-injected controls (P < 0.005). For peripheral blood studies, turkeys and chicks were treated as above, and at 4 hr post-PBS, ILK, or VILK injection; total and differential peripheral blood counts were performed on birds from each group. A significant (P < 0.05) peripheral blood heterophilia at 4 hr postinjection was observed in the ILK- and VILK-treated birds, with no such increase found in the PBS-injected group. Correlation analysis revealed a direct relationship between the peripheral blood heterophilia in turkeys and chicks seen at 4 hr postinjection and the reduction in SE organ invasion seen in the VILK and ILK treatment groups (r = 0.991, r = 0.91, respectively). T cells isolated and transformed from nonimmune chickens did not produce factors that protected chicks from SE organ invasion and did not cause the peripheral blood heterophilia observed with ILK and VILK. These results show that the virally transformed SE-immune T-cell line produces lymphokines that result in the same level of peripheral blood heterophilia as ILK and was equally protective against SE organ invasion as ILK.

Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T.

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.

The N-terminal env-derived amino acids of v-rel are required for full transforming activity

Expression of the v-rel oncogene of the reticuloendotheliosis virus, strain T (REV-T), can mediate the transformation of chicken spleen and bone marrow cells. Although the majority of the coding sequence of the v-rel oncogene is derived from the cellular rel sequence, the N- and C-terminal amino acids are coded for by remnants of the REV env gene. The resulting v-rel protein can be described as an env-rel-(out of frame env) fusion protein. Terminal deletion mutants were constructed to determine the role that env sequences play in the transforming activity of v-rel. Deletions were designed to remove only sequences of v-rel derived from former env sequence. Additional deletions removed more substantial amounts of coding sequence. Introduction of deleted genes into an REV-T based retroviral vector permitted the transforming activities to be determined. Deletion analysis indicated that the N-terminal region of pp59 v-rel is required for the transforming activity, whereas as many as 100 C-terminal amino acids could be deleted without complete loss of the activity.

Propagation of reticuloendotheliosis virus strain t in cell lines derived from avian lymphoid leukosis lymphoma and Marek's disease tumour

Propagation of reticuloendotheliosis virus strain T (REV-T) was studied in avian lymphoid leukosis lymphoma-derived cell lines (LSCC-1104x5 and BK 3) and Marek's disease tumour-derived cell lines (MDCC-MSB1 and RP 1). These four cell lines showed cellular growth for a long time even after they were infected with REV-T. Production of non-oncogenic REV-T, replication non-defective virus, in these cell lines continued for 3 to 7 months. When culture fluids from 1104x5, MSB1 and RP 1, which were infected with oncogenic REV-T, were inoculated into 1-day-old chickens, acute development of lethal reticuloendotheliosis (RE) tumours occurred. Especially after the use of the fluids from 1104x5, propagation of oncogenic REV-T was observed for a long time, 7 months. Of the chickens inoculated with the culture fluid of any REV-T-infected cell lines, many chickens which survived for 4 weeks showed abnormal feathering, 'nakanuke'. The body weight of these chickens was significantly less than that of uninoculated chickens. No contamination with infectious REV was observed in the culture fluids from any uninfected line cells.

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.

Autogenous growth factor production by reticuloendotheliosis virus-transformed hematopoietic cells.

Reticuloendotheliosis virus strain T (REV-T)-transformed cells gave rise spontaneously to variants which secrete a factor that forms a distinct visible ring of precipitation (halo) surrounding colonies grown in soft agar. An Mr 15,000 protein was produced at higher levels by halo variants than by nonhalo-producing cells. An assay designed to detect the formation of precipitates enabled purification of an Mr 15,000 protein, p15, from serum-free medium conditioned by the growth of REV-T-transformed hematopoietic cells. Fractions enriched in p15 permitted the growth of REV-T-transformed cells under conditions where they normally failed to proliferate.

Localization of the v-rel protein in reticuloendotheliosis virus strain T-transformed lymphoid cells.

The protein (p59rel) encoded by the transforming gene of reticuloendotheliosis virus strain T (REV-T) has been identified in REV-T-transformed avian lymphoid cells by using antisera raised against synthetic peptides whose sequences were derived from three nonoverlapping regions of v-rel (N. R. Rice, T. D. Copeland, S. Simek, S. Oroszlan, and R. V. Gilden, Virology 149:217-229, 1986). To obtain polyclonal antibodies directed against a larger number of p59rel epitopes, a 262-amino acid segment was expressed in bacteria. Antisera raised against this fusion protein (v-delta-rel) precipitated p59rel from lysates of [35S]methionine-labeled REV-T-transformed cells, thus confirming previous results obtained with the peptide antisera. We used this new antiserum to localize p59rel in REV-T-transformed cells by subcellular fractionation using differential centrifugation and by indirect immune fluorescent staining. After fractionation and immune precipitation, the majority of p59rel was found in the cytosolic fraction. Indirect immunofluorescence experiments also gave results consistent with the cytoplasmic localization of the v-rel protein in transformed lymphoid cells. In previous studies (Rice et al., Virology 149:217-229, 1986) it was shown that immune precipitates formed with one of the three p59rel peptide antisera possessed in vitro protein kinase activity. Immune precipitates formed with the fusion protein antiserum also showed kinase activity in the in vitro assay. Most of this activity was found in the soluble cytoplasmic fraction, indicating that the kinase may be p59rel or a protein closely associated with it.

Different localization of the product of the v- rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus that transforms early lymphoid cells in vivo and in vitro, but REV-T does not transform chicken embryo fibroblasts (CEF). Using antisera to p59 v- rel , the v- rel oncogene product of REV-T, we show that p59 v- rel is expressed at equal levels and is a phosphoprotein in REV-T infected spleen cells and CEF. Biochemical fractionation and immunofluorescence of REV-T infected nontransformed CEF show that p59 v- rel is loosely associated with the nucleus. However, in REV-T transformed spleen cells p59 v- rel is primarily a cytoplasmic protein. MSB-1 cells, a Marek's disease virus transformed T cell leukemic line, and E26 virus transformed myeloid cells show nuclear staining of p59 v- rel when they are infected by REV-T. Our results indicate that there is a correlation between a cytoplasmic localization of p59 v- rel and transformation by REV-T, and they suggest that p59 v- rel cannot transform cells in which it assumes solely a nuclear location.

Identification of the v-rel protein in REV-T transformed chicken bone marrow cells and expression in cosi cells

The product of the v-rel oncogene has been identified as a 55,000 dalton protein using antiserum prepared against a synthetic polypeptide whose sequence was deduced from the DNA sequence of v-rel. This antiserum was used in a Western blot assay to identify the product of v-rel in chicken bone marrow cells transformed with reticuloendotheliosis virus, strain T (REV-T), and in Cos1 cells transfected with an expression vector containing the v-rel gene. Transient expression of v-rel under the transcriptional control of the SV40 late promoter in Cos1 cells leads to the synthesis of a rel specific protein with a similar molecular weight.

Characterization of reticuloendotheliosis virus strain T DNA and isolation of a novel variant of reticuloendotheliosis virus strain T by molecular cloning.

Reticuloendotheliosis virus strain T (REV-T) is a highly oncogenic avian retrovirus which causes a rapid neoplastic disease of the lymphoreticular system. Upon infection, this virus gives rise to two species of unintegrated linear viral DNA, which are 8.3 and 5.5 kilobase pairs long and represent the helper virus (REV-A) and the oncogenic component (REV-T), respectively. Restriction endonuclease cleavage maps of these two DNA components indicate that REV-T DNA has a large portion of the genome deleted with respect to REV-A DNA and a substitution about 0.8 to 1.5 kilobase pairs long that is unrelated to REV-A DNA. These additional sequences comprise the putative transforming region of REV-T (rel). A chicken spleen cell line transformed by REV-T produced virus which upon infection gives rise to three species of unintegrated linear viral DNA (8.3, 5.5, and 3,3 kilobase pairs). We isolated the proviruses of the 8.3- and 3.3-kilobase pair species from this cell line by cloning in the phage vector Charon 4A. Restriction enzyme mapping showed that the two proviral clones are proviruses of REV-A and a variant of REV-T, respectively. A subclone of the variant REV-T provirus specific for the rel sequences of REV-T was used as a hybridization probe to demonstrate that the rel sequences are different from the putative transforming sequences of Schmidt-Ruppin Rous sarcoma virus strain A, avain myelocytomatosis virus, avian myeloblastosis virus, avian erythroblastosis virus, Abelson murine leukemia virus, and Friend erythroleukemia virus. In addition, the rel-specific hybridization probe was used to identify a specific set of sequences which are present in uninfected avian DNAs digested with several restriction enzymes. The corresponding cell sequences are not arranged like rel in REV-T.

Continuous cell culture from chick embryos inoculated with rev strain t

Oncogenic reticuloendotheliosis virus strain T (REV-T) was inoculated into 9-day-old embryonated eggs of specific pathogen free chickens. A continuous cell line was established from the liver cells of one embryo harvested the day before hatching which had the following properties: It formed loose aggregates and propagated floating in the medium. The cell doubling time was about 24 hours. Viral antigen was demonstrated in the cytoplasm by the direct immunofluorescent antibody technique. C-type particles were observed outside the cells. Virus-induced cell surface antigen was demonstrated by the immunofluorescent antibody technique. The cell line grew in soft agar. Virus production continued in the cell line for 17 months. When the cells or the culture fluid were inoculated into 1-day-old chickens, they died from neoplastic disease. The chickens which survived after infection showed abnormal feathering ("nakanuke"). The virus released from this cell-line was inoculated into 9-day-old embryonated eggs and three cell lines were established. These cell lines did not contain avian leukosis/sarcoma viruses.

A Survey of Non-Specific Cross-Protective Immunities Induced by Avian Retroviruses

Reticuloendotheliosis virus strain T (REV-T) induced immunity in chicks to challenge by representative subgroup members of the avian leukosis-sarcoma virus (ALSV) complex. Immunity levels were compared to determine the extent of antigenic relation between REV-T and the ALSV complex. Reciprocal studies using ALSV subgroup members and pheasant viruses as immunogens and REV-T as challenge were also performed. It was concluded that reciprocity of immunity is not equal between the viruses studied, nor is immunity directly related to the virus-neutralizing-antibody levels induced by immunization with the viruses studied. In some cases, the levels of cross-protection demonstrated may be a sign of the induction of antibodies to common or similar tumor-specific surface antigens rather than complete antigenic identity between REV-T and the ALSV members used; in others, virus-neutralizing antibodies may be a sign of partial identity between some proteins of REV-T and ALSV subgroup members.

Avian reticuloendotheliosis virus: characterization of the high-molecular-weight viral RNA in transforming and helper virus populations.

Reticuloendotheliosis virus strain T (REV-T) is a type C retrovirus known to transform avian fibroblasts, spleen cells, and bone marrow cells and to produce virulent reticuloendotheliosis in young chicks. Analysis of REV-T high-molecular-weight RNA by electrophoresis in denaturing gels and by electron microscopy revealed the presence of at least two classes of molecules. One class appeared in CH3HgOH gels to have a monomer length of 9.3 kilobases (kb); in electron microscopic spreads under mildly denaturing conditions, it existed as a typical retrovirus dimer, having a monomer length of 8.8 +/- 0.7 kb. The second class also existed as a dimer, with a monomer length of 5.7 kb in CH3HgOH gels. Hybridization with REVA-A 32P-labeled complementary DNA revealed a third size class of molecules (4.7 kb), which were not resolvable from the 5.7-kb class in electron microscope spreads and which comigrated with chicken 28S rRNA in denaturing gels. Only the 9.3-kb class was found in the reportedly nontransforming virus produced after infection of canine thymus cell line with REV-T. Thus, REV-T appears to be similar to the murine and feline sarcoma viruses and the avian acute leukemia viruses in that it consists of a nontransforming helper virus genome and a defective genome responsible for oncogenicity. Our previous results demonstrated the presence in REV-T and in uninfected chicken cellular DNA of some nucleotide sequences not found in virus produced by the canine line (S. Simek and N. Rice, J. Virol. 33:320--329, 1980). In this report we show by hybridization with highly specific 32P-labeled complementary DNAs that REV-T-specific sequences exist within the 5.7-kb genome. Since 32P-labeled complementary DNA synthesized from the canine-derived virus genome hybridized with all three classes of RNAs, we conclude that the 5.7-kb genome is a recombinant between some sequences found in the putative helper and some sequences specific to REV-T. As with the other oncogenic viruses mentioned above, these specific sequences appear to be derived from host DNA.