Deletions in one domain of the Friend virus-encoded membrane glycoprotein overcome host range restrictions for erythroleukemia.

Although the Friend virus-encoded membrane glycoprotein (gp55) activates erythropoietin receptors (EpoR) to cause erythroblastosis only in certain inbred strains of mice but not in other species, mutant viruses can overcome aspects of mouse resistance. Thus, mice homozygous for the resistance allele of the Fv-2 gene are unaffected by gp55 but are susceptible to mutant glycoproteins that have partial deletions in their ecotropic domains. These and other results have suggested that proteins coded for by polymorphic Fv-2 alleles might directly or indirectly interact with EpoR and that changes in gp55 can overcome this defense. A new viral mutant with an exceptionally large deletion in its ecotropic domain is now also shown to overcome Fv-2rr resistance. In all cases, the glycoproteins that activate EpoR are processed to cell surfaces as disulfide-bonded dimers. To initiate analysis of nonmurine resistances, we expressed human EpoR and mouse EpoR in the interleukin 3-dependent mouse cell line BaF3 and compared the abilities of Friend virus-encoded glycoproteins to convert these cells to growth factor independence. Human EpoR was activated in these cells by erythropoietin but was resistant to gp55. However, human EpoR was efficiently activated in these cells by the same viral mutants that overcome Fv-2rr resistance in mice. By construction and analysis of human-mouse EpoR chimeras, we obtained evidence that the cytosolic domain of human EpoR contributes to its resistance to gp55 and that this resistance is mediated by accessory cellular factors. Aspects of host resistance in both murine and nonmurine species are targeted specifically against the ecotropic domain of gp55.

Isolation and characterization of an interleukin-8-like peptide in the bovine species.

Human interleukin-8 (IL-8) is a biologically active peptide which displays chemo-attractive activity for neutrophils and T-cells. The molecule is produced by a variety of cell types upon exposure to lipopolysaccharide, interleukin-1 and tumor necrosis factor. Recombinant human IL-8 also stimulates chemotaxis of bovine cells in a dose dependent manner. The purpose of this series of studies was to investigate the ability of bovine cells to produce an active IL-8-like molecule and to determine if bovine cells respond to human recombinant IL-8. Stimulation of purified peripheral blood mononuclear cells results in the time dependent production of an IL-8-like molecule as determined using an anti-human IL-8 ELISA assay and a bovine neutrophil chemotactic assay. Physical characterization indicates that the biological activity of the molecule was significantly reduced by heat inactivation at 56 degrees C for 30 min or exposure to extreme acidic or basic conditions. The peptide was affinity purified using an anti-human IL-8 antibody produced from ATCC hybridoma HB9647. SDS-PAGE analysis yields a distinct band at 7.8 kDa. The isoelectric point of the purified protein was determined to be 8.65. Biological activity of the purified protein was confirmed and the anti-human IL-8 antibody was capable of partially neutralizing the chemotactic activity.

Rapid capping in alpha-spectrin-deficient MEL cells from mice afflicted with hereditary hemolytic anemia.

A spectrin-based membrane skeleton is important for the stability and organization of the erythrocyte. To study the role of spectrin in cells that possess complex cytoskeletons, we have generated alpha-spectrin-deficient erythroleukemia cell lines from sph/sph mice. These cells contain beta-spectrin, but lack alpha-spectrin as determined by immunoblot and Northern blot analyses. The effects of alpha-spectrin deficiency are apparent in the cells' irregular shape and fragility in culture. Capping of membrane glycoproteins by fluorescent lectin or antibodies occurs more rapidly in sph/sph than in wild-type erythroleukemia cells, and the caps appear more concentrated. The data support the idea that spectrin plays an important role in organizing membrane structure and limiting the lateral mobility of integral membrane glycoproteins in cells other than mature erythrocytes.

A Friend virus mutant that overcomes Fv-2rr host resistance encodes a small glycoprotein that dimerizes, is processed to cell surfaces, and specifically activates erythropoietin receptors.

The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger dimeric plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. D. D'Andrea, H. F. Lodish, and D. Baltimore, Nature (London) 343:762-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-2r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, D. Kabat, and R. W. Geib, J. Virol. 66:3652-3660, 1992). Mutant BB6, which encodes a gp42 glycoprotein that has a large deletion in this domain, causes erythroblastosis in DBA/2 (Fv-2s) as well as in congenic D2.R (Fv-2r) mice. Analogous to gp55, gp42 is processed inefficiently as a disulfide-bonded dimer to form cell surface gp42p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 125I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 125I-Epo-gp55p and 125I-Epo-gp42p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue. Our results indicate that the SFFV and BB6 env glycoproteins specifically activate EpoR; they help to define the glycoprotein properties important for its functions; and they strongly suggest that the Fv-2 leukemia control gene encodes an EpoR-associated regulatory factor.

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor.

RB virus: a strain of Friend virus that produces a 'Friend virus-like' disease in Fv-2rr mice.

RB virus is a newly derived strain of Friend virus that was adapted to produce a 'Friend-like' disease in mice that are genetically resistant to wild-type Friend virus. RB virus was produced by passing high titers of the wild-type Friend virus (Lilly-Steeves polycythemia-producing strain) through adult Fv-2rr mice. Titration of the defective spleen focus-forming virus indicated RB virus infected similar numbers of Fv-2ss or Fv-2rr target cells. Analysis of the spleens from mice infected with RB virus indicated that RB induced the early stage of Friend disease (erythroid proliferation) in both Fv-2rr and Fv-2ss mice. Fv-2ss mice infected with RB virus developed the classical Friend disease within 3 weeks. In contrast, the percentage of Fv-2rr mice developing the 'Friend-like' disease after infection with RB virus never exceeded 60%. The latency period of RBV in Fv-2rr mice was strain dependent. D2.R16 (Fv-2rr) developed the syndrome more rapidly than C57BL/6 (Fv-2rr). RB virus retained the capacity to transform erythroprogenitor cells from both Fv-2ss and Fv-2rr animals. Cells infected with RB virus consistently produced a modified SFFV envelope protein, gp48.

Fine mapping of an H-2Kk restricted cytotoxic T lymphocyte epitope in SV40 T antigen by using in-frame deletion mutants and a synthetic peptide.

The CTL response to SV40 in C3H/HeJ mice is directed against the tumor (T) Ag and is H-2Kk restricted. CTL specific for both the amino terminus (residues 1-271) and the carboxyl terminus (residues 512-708) of the T Ag molecule have been detected, and we have previously cloned CTL of both specificities. In this paper we show that the panel of 10 CTL clones specific for the C-terminal region includes clones specific for three different epitopes, termed C1, C2, and C3. Epitopes C1 and C2 are conserved in the T Ag of the related papova viruses BK and SA12, and only epitopes C2 and C3 are present on SV40 transformed targets bearing the Kk mutant Kkml. Epitopes C1 and C2 were mapped to residues 563-576 by using in-frame deletion mutants of SV40 T antigen, and all clones specific for these two epitopes can lyse Kk bearing target cells in the presence of a synthetic peptide comprising residues 559-576. Kk and Kkml differ at residue 152, which is located in the Ag-binding pocket. Because epitopes C1 and C2 can be formed by the same antigenic peptide, but epitope C1 is not present on SV40 transformed Kkml cells, epitopes C1 and C2 must differ in the contribution made by residue 152 of the MHC class I molecule. These data show that CTL epitopes on transformed cells can be made up of Ag fragments, and strengthen the idea that this is a general phenomenon for both class I and class II restricted T cell epitopes.

Inhibition of Nb2 T-lymphoma cell growth by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) inhibits proliferation of Nb2 cells, a rat T lymphoma, in response to lactogens and interleukin-2. Prostaglandins may play an important role in the pathway through which TGF-beta exerts its inhibitory actions, because prostaglandin E2 also inhibits proliferation of Nb2 cells, and indomethacin, an inhibitor of prostaglandin synthesis, reverses the inhibitory effects of TGF-beta on Nb2 cell proliferation.

Cellular control of in vitro progression of murine myeloid leukemia: progression accompanies acquisition of independence from growth factor and stromal cells.

The ability of bone marrow stroma cells of normal WCB6F1 (+/+) mice versus their congenic Sl/Sld stromal-defective littermates to support sustained proliferation and leukemic transformation of the growth factor-dependent myeloid cell line FDC-P1 was studied. Extensive proliferation of factor-dependent cells occurred on (+/+) normal long-term marrow culture stroma without the addition of growth factor, whereas factor-dependent cells dissipated from Sl/Sld stromal cultures after addition. The sustained proliferation that occurred on +/+ stromal layers later resulted in the appearance of factor-independent cell lines that were no longer dependent upon stroma. Factor-independent cell lines were cloned by limiting dilution and analyzed for expression of cell surface antigens to prove their origin from FDC-P1. Factor-independent cells, but not factor-dependent cells, formed tumors in syngeneic mice. These studies demonstrate a critical role for marrow stroma in the stepwise development of murine leukemia and are concordant with the previous data obtained in in vivo studies by McCool et al. that the splenic stroma of irradiated Sl/Sld mice do not support growth of Friend virus-induced preleukemic cell colonies. The present data demonstrate in a preleukemia model not induced by Friend virus complex that normal (+/+) stromal cells promote the in vitro proliferation of factor-dependent preleukemic cells and their subsequent transition to factor-independent leukemia cells, but Sl/Sld defective stroma do not efficiently promote this transition.

Infection and transformation of Fv-2rr erythroprogenitor cells with Friend virus.

Friend virus was used to infect and transform Fv-2rr erythroprogenitor cells in vivo. The RB (Fv-2rr) cell line was characteristic of Friend virus-induced cell lines in Fv-2ss mice, i.e., it produced infectious Friend virus and synthesized hemoglogin. The RB (Fv-2rr) cell line expressed the envelope protein of the spleen focus-forming virus (gp52) and a novel, related envelope protein (gp48). The results demonstrate that Fv-2rr erythroprogenitor cells can be infected and transformed in vivo.

Characterization of cell lines derived from enlarged spleens induced in C57BL/6 mice by the variant BSB strain of Friend erythroleukemia virus.

BSB is a variant strain of Friend virus selected for pathogenicity in C57BL/6 mice that are resistant to parental Friend virus strains by virtue of their homozygosity for the recessive Fv-2r allele (Steeves et al., 1970, Int. J. Cancer 5, 346-356). Lines and clones of erythroleukemia cells could readily be established in culture from the enlarged spleens of BSB-infected Fv-2r homozygotes. All lines expressed viral gene products and could be induced to express hemoglobin. Some lines produced infectious virus. In addition to the viral envelope-related proteins (gPr85, gp70, and gp52) detected by precipitation with goat anti-Rauscher gp70 antiserum from tumor cell lines induced by parental Friend virus strains, BSB-induced cell lines also expressed gp80, p52, and gp45 products precipitable with the same antiserum. A rat monoclonal antibody that recognizes an epitope of an amino-terminal region of gp52 (Wolff et al., 1982, J. Virol. 43, 472-481) also precipitated the gp80 and gp45 viral proteins. The data indicate that the BSB strain of Friend virus is oncogenic in Fv-2r homozygotes. Transformation is correlated with the expression of an altered SFFV env-gene product.

MLR blast cells generated in mutant-standard strain combinations bind H-2K and H-2D antigens.

Strains CBA (M523) (= M523) and CBA differ by a mutation which has been mapped genetically into the K region of the H-2 complex. Similarly, strains B 10.D2 (M504 (= M504) and b 10.D2 differ in a mutation which occurred in the D region. The data presented in this study show that mixed lymphocyte culture in M523 anti-CBA and M504 anti-B 10.D2 strain combinations leads to the release of membrane fragments from the stimulating cells and binding of these fragments by blast cells. The fragments always carry H-2K (in the M523 anti-CBA combination) or H-2D (in the M504 anti-B 10.D2 combination) antigens present in the stimulating and absent in the responding cells (antigens H-2.60 and H-2.40, respectively). Although Ia antigens may occasionally be present on the CBA membrane fragments, these antigens do not participate in the M523 anti-CBA MLR stimulation. The data thus demonstrate that serologically detectable H-2K and H-2D antigens can induce a MLR and that a mutation can change properties of H-2K or H-2D molecules so that the alteration is detectable by both serological means and lymphocyte activation assays.