Friend Virus Infection Research Articles

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Cellular expression of murine leukemia virus gp70-related antigen on thymocytes of uninfected mice correlates with Fv-4 gene-controlled resistance to friend leukemia virus infection

Alleles at the Fv-4 locus have been shown to determine susceptibility ( Fv-4 s ) and resistance ( Fv-4 r ) to infection with ecotropic murine leukemia viruses (MuLV). The resistance is dominant in heterozygous mice. BALB/c mice are Fv-4 ss and a semicongenic line BALB/c- Fv-4w r , homozygous for Fv-4 r allele and called C4W, is being developed. Extracts of 125I-labeled thymocytes from BALB/c and C4W mice were precipitated by a series of antisera against MuLV and the precipitates examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Thymocytes from C4W mice expressed 80K and 73K glycoproteins related to the gp70 of MuLV, while only the 73K glycoprotein was occasionally detectable on BALB/c thymocytes. Genetic studies showed that a locus controlling expression of the 80K protein was dominant and associated with the resistant allele at Fv-4. This membrane gp70 may be important in the Fv-4-mediated resistance to exogenous virus infection.

Use of H-2:H-7 congenic mice to study H-2-mediated resistance to Friend leukemia virus.

Congenic and double congenic mice expressing different H-7 alleles with varying H-2 haplotypes on the C57BL/10 (B10) background were tested for their susceptibility to Friend virus (FV) infection. Mice expressing the H-7b allele derived from BALB/c were susceptible to FV infection whereas mice expressing the H-7a allele of B10 were absolutely resistant. Coexpression of H-7b and the H-2a and H-2d haplotypes determined high susceptibility; mice expressing both H-7b and H-2b were relatively resistant compared to H-7b mice expressing H-2a or H-2d. Parallel experiments with BALB/c and BALB.B recipients did not demonstrate differences in susceptibility associated with H-2d and H-2b. Mapping experiments with the H-2h4 and H-2i5 recombinants indicated that the gene determining relative resistance to FV-induced spleen focus formation mapped to the K-end (KbAb) of H-2b. Female recipients expressing H-7b, regardless of their H-2 haplotype, were more susceptible to FV infection than their syngeneic male counterparts. These experiments demonstrate the utility of congenic and double congenic strains that define H-7 and Fv-2 on different H-2 haplotype backgrounds for the analysis of Fv-2:H-7:H-2 interactions in determining susceptibility to FV infection.

Impairment of in vitro generation of cytotoxic or T suppressor lymphocytes by Friend leukemia virus infection in mice.

Spleen cells collected from DBA/2 (H-2d) mice inoculated with the polycythemic variant of Friend-Leukemia Virus Complex (FLV-P) were tested for T-dependent immune functions, such as the in vitro generation of cytotoxic T lymphocytes (CTL) and of non-specific T suppressor lymphocytes (STL). CTL were generated against H-2b splenocytes, and STL were obtained following a 5-day lymphocyte culture without stimulator cells. A progressive and severe impairment of the generation of both CLT and STL was found from 2 weeks onward after infection, being almost totally abolished 3-4 weeks after virus challenge. Suppressor cells (SC) capable of inhibiting CTL generation was detected in FLV-P bearing mice. Suppressor activity was unaffected by anti-Thy 1.2 serum and complement but was removed following iron-magnet depletion or passage through nylon-wool column. Moreover complete recovery of the competence of CTL generation was attained when FLV-P infected splenocytes were passed through nylon-wool column. It is concluded that FLV-P infection depresses T-dependent cytotoxic and suppressor responses in mice, by the appearance of non-T adherent phagocytic cells, capable of impairing CTL generation in vitro.

New cell surface gp70 related to Friend mink cell focus-inducing virus is expressed on Friend virus-induced erythroleukemic spleen cells after elimination of ecotropic Friend virus gp70 in Rfv-3r/s mice.

Spleen cells from Rfv-3r/s mice with Friend virus-induced erythroleukemia were analyzed for expression of virus-induced proteins with monoclonal antiviral antibodies and conventional antisera. Leukemic spleen cells, 30-60 d after virus inoculation, expressed decreased amounts of ecotropic Friend murine leukemia helper virus gag- and env-encoded cell surface and intracellular proteins compared with leukemic cells tested 8-10 d after virus inoculation. In contrast, the spleen focus-forming virus-induced protein, gp55, was present on both leukemia cell populations. This difference appeared to be mediated by the humoral antibody response in Rfv-3r/s mice, which could recognize only ecotropic gag and env proteins, and not gp55. A new gp70 molecule cross-reactive with a recombinant Friend mink cell focus-inducing virus was found in large quantities on late leukemic cells. This protein appeared to be derived from a recombinant virus produced during the course of Friend virus infection. The appearance of this new gp70 suggests that recombinant viruses other than spleen focus-forming virus may play a role in Friend virus-induced erythroleukemia.

Effect of anti-interferon serum on Friend virus infection and Fv-1-mediated resistance.

The purpose of these studies was (1) the quantitation of the protective effect of endogenous interferon (IFN) in the Friend virus (FV) system, and (2) the determination of the role of IFN in the relative resistance to FV mediated by the Fv-1-gene. In these studies, mice from two strains differing with respect to their Fv-1 alleles were treated with sheep anti-mouse IFN serum immediately prior to infection with N-tropic FV. The effect of this pretreatment showed that the neutralization of FV-induced IFN by anti-IFN resulted in a two to tenfold increase in susceptibility to FV infection as measured by the appearance of macroscopic foci of erythroleukemic cells on the splenic surface as compared to control mice which received normal sheep serum. At higher virus doses, an increase in spleen weight in anti-IFN-treated mice was also observed. Mice resistant to FV by virtue of their Fv-1 allele were affected by anti-IFN pretreatment only as much as mice from the susceptible strain. Therefore, IFN appeared to play no role in Fv-1-mediated resistance.

Effect of serum from mice with dormant Friend leukemia viral infections on synthesis and modulation of erythroleukemia cell surface gp70.

Serum of mice with dormant Friend virus infections modulates the expression of cell surface antigens on Friend virus-transformed cells (FLC-745 cells). By radioimmunoassay, the modulated antigen was identified as virion gp70 of Friend leukemia virus. After two days of culture in medium containing 5% serum from mice with dormant Friend virus infections, the modulated cells expressed about 50% less surface gp70 than non-modulated cells cultured in 5% normal mouse serum. After 9 days in culture, the modulated cells expressed no surface gp70; however, these cells contained the same amount of internal gp70 as nonmodulated cells, indicating that gp70 was synthesized during the modulation period. Both modulated and nonmodulated cells grew equally well during the 9 days of culture. These results indicate that the effects of modulation of Friend virus-coded cell surface antigens are restricted to the cell surface.

Anti-Friend virus antibody is associated with recovery from viremia and loss of viral leukemia cell-surface antigens in leukemic mice. Identification of Rfv-3 as a gene locus influencing antibody production.

A single genetic locus, Rfv-3, influenced Friend virus (FV) viremia, loss of FV-induced cell-surface antigens from leukemia cells, and generation of anti-FV antibodies. 30--90 d after FV infection leukemic spleen cells from (B10.A X A)F1 and (B10.A X A.BY)F1 mice (Rfv-3r/s) were found to have low FV-induced cell-surface antigen expression compared to leukemic spleen cells from A and A.BY mice (Rfv-3s/s). In addition, these F1 mice recovered from viremia and generated cytotoxic anti-FV antibodies. A and A.BY mice did not recover from viremia and failed to generate anti-FV antibodies. Anti-FV leukemia cell antibody appeared to mediate FV-antigen loss because decrease of FV cell-surface antigens occurred at the same time as anti-FV antibody appeared in the plasma of F1 mice, and passive transfer of anti-FV antisera induced modulation of FV cell-surface antigens. Rfv-3 did not influence an intrinsic ability of FV antigens to be modulated from Rfv-3s/s leukemia cells because FV antigen loss from Rfv-3s/s spleen cells occurred after transfer of cells to an immune environment.

Susceptibility of Friend Virus Antigen-Modulated Erythroleukemic Cells to Lysis by T Lymphocytes from Mice with Dormant Friend Virus Infections

Spleen cells from DBA/2 mice with dormant Friend leukemia virus (FLV) infections or from mice immunized with x-irradiated FLC-745 erythroleukemic cells are not cytolytic for FLC-745 cells when tested directly, but acquire cytolytic activity in vitro by cultivation with x-irradiated FLC-745 cells. Spleen cells cultured from normal mice do not acquire such cytolytic activity. Cytolytic activity resides in the T lymphocyte population. Alloantiserum, but not antisera against FLV-virion polypeptides, inhibited the lysis of FLC-745 cells by cytolytic T lymphocytes. To understand further the role of cellular and humoral immune anti-FLV responses in mice with dormant FLV-infections, in vitro experiments were conducted that mimicked the in vivo FLV-specific immune environment of these mice. We found that FLV-immune serum from mice with dormant FLV-infections modulated FLV-antigen expression on the surfaces of FLC-745 cells without affecting their susceptibility to lysis by cytolytic T lymphocytes. These results suggest that cytolytic T lymphocyte restrain the outgrowth of FLV-antigen-modulated erythroleukemic cells in mice with dormant FLV infections and that the targets for cell-mediated lysis may be H-2d-associated antigens.

Splenic erythroid response to friend polycythemia virus: time course in vitro after infection in vivo.

When spleen cells removed from plethoric BALB/c mice shortly after infection with Friend polycythemia virus were cultured, they subsequently increased their rate of hemoglobin synthesis in vitro without the addition of the hormone erythropoietin. The increased 59Fe incorporation into hemoglobin in vitro was part of a well-defined single wave unlike the progressive increase in hemoglobin synthesis that occurs in vivo. The peak occurred within the same total time (85-105 hr after infection), irrespective of either the virus dose or time after infection when the cells were removed from the animal and cultured. However, the magnitude of the peak increased with an increase in either of these two variables. Medium change experiments indicated that the time during which the peak occurred was not artificially determined by depletion of some medium component or the accumulation of an inhibitor. This system may be useful in separating the early events of Friend virus infection from the late effects on erythroid differentiation.

The Fv-2r resistance gene in mice: its effect on spleen colony formation by Friend virus-transformed cells.

To determine the mechanism by which the recessive Fv-2r gene confers complete resistance to spleen focus formation and to the induction of early splenomegaly characteristic of the Friend virus (FV) disease syndrome, we compared the parameters of FV infection in susceptible DBA/2 (D2) (Fv-1n, Fv-2s) and partially congenic D2.Fv-2r (Fv-1n, Fv-2r) mice. After infecting these mice with N-tropic FV complex, we followed the replication of spleen focus-forming virus (SFFV) and the generation of SFFV-transformed tumor colony-forming cells (CFC) in their spleens. By both parameters D2.Fv-2r mice were 20- to 100-fold less susceptible than DBA/2 controls, but the inhibition was only partial. Transplantation of washed spleen cells from SFFV-infected DBA/2 mice resulted in equal growth and recovery of tumor CFC from both D2.Fv-2r and Fv-2s mice. However, these tumor cells grew as colonies in Fv-2s mice, whereas they grew diffusely in the spleens of D2.Fv-2r hosts and did not develop into macroscopically or microscopically visible colonies. Thus the completeness of the resistance to spleen focus formation that defines the Fv-2r gene was reflected only in the complete suppression of the colonial growth of tumor cells, whereas the other parameters of infection showed no or only partial inhibition.

The spleen in Friend leukemia. I. Prolonged survival of leukemic mice after autoimplantation of spleen tissue.

Friend virus infection of susceptible mice led rapidly to fulminant erythroleukemia and death. Subcutaneous implantation of leukemia spleen bits into splenectomized normal animals led to their early death from Friend leukemia. In contrast, bits of leukemic spleen implanted sc into splenectomized leukemic mice prolonged the survival of these animals. Concomitant with this survival was a reversal of the virus-induced immunosuppression and an increase in the levels of circulating, neutralizing, antivirus activity. This marked difference in response to leukemic spleen implants by leukemic as compared to normal mice reflected previous contact of the former with Friend Virus. Our studies indicated that the Friend virus-infected mouse mounted a resistance to the virus infection, which under certain conditions is capable of reversing the disease process.

In vitro depression of cellular immunity by friend virus leukemic spleen cells

Murine spleen lymphocytes show a marked depression in their response to concanavalin A (Con A) 14 days after Friend virus (FV) infection. This depression is related to the appearance of FV antigen-containing cells in the spleen. Statolon treatment 3 days after FV inoculation suppresses FV disease and restores the responsiveness of lymphocytes to Con A. The addition of as few as 1 leukemic to every 5 normal spleen cells inhibits the Con A response of the latter cells. Normal spleen lymphocytes are not inhibited by the addition of FV or leukemic spleen cell culture fluid. Leukemic cells have an increased avidity for Con A and preferentially remove it from the culture medium. However, increasing the concentration of Con A in the medium did not overcome the depressive effects of the leukemic spleen cells on normal cells, indicating that depression of the normal cellular response to Con A had been produced by the leukemic cells. The data suggest that this depressive effect is mediated via membrane-membrane contact between the two cell types.

Erythropoietin-independent erythroid colony formation in vitro by hemopoietic cells of mice infected with friend virus.

We have investigated the production of erythroid colonies in plasma culture by bone-marrow and spleen cells taken form C3Hf/Bi mice previously infected with a polycythemic strain of Friend virus (FV). Inclusion of erythropoietin (Epo) in the medium was found unnecessary for erythroid colony formation in vitro by these cells, although it was essential for the production of erythroid colonies by hemopoietic cells from normal animals. Development of erythroid colonies also proceeded umimpeded when cells from FV-infected animals were cultivated in medium pretreated with rabbit anti-serum that was shown to inactivate Epo. Thus, the hemopoietic tissues of FV-infected mice contained erythroid colony-forming units (CFU-Es) which appeared to be Epo-independent. When spleen cells from FV-infected mice were exposed to antiserum directed against syngeneic FV-infected spleen cells and complement, and then cultured with or without Epo, the number of erythroid colonies that developed was drastically reduced, indicating that the CFU-Es in these animals carried FV-induced antigen(s), and must themselves have been infected with virus. Electron microscopy of erythroid colonies produced by cells from FV-infected mice revealed the presence of budding and abundant free type-C virus particles. The efficiency of erythroid colony formation in vitro either with or without Epo by hemopoietic cells from FV-infected mice was substantially increased over that of cells from normal mice. The increase in the number of CFU-Es in these animals was due mainly to an increase in the number of Epo-independent CFU-Es. Epo-independent CFU-Es were first detected in bone marrow and spleen as early as 3 days after FV infection; thereafter their numbers progressively increased for at least 9 days. Hypertransfusion with red blood cells prior to FV infection reduced, while bleeding greatly increased, the efficiency of erythoid colony formation without Epo by cells from the spleens of the infected mice. The phenomenon of erythroid colony formation in plasma cultures lacking Epo provides a sensitive and reliable means of detecting Epo-independent CFU-Es, which appear to play a fundamental part in pathogenesis of the disease resulting from infection with the polycythemic strain of FV.

MECHANISMS OF GENETIC RESISTANCE TO FRIEND VIRUS LEUKEMIA IN MICE

Resistance to malignant erythropoiesis induced by Friend spleen focus-forming virus and resistance to marrow stem cell allografts are under genetic control. Strains of mice, e.g., C57BL/6 and B10.D2, which are homozygous for resistance at the Fv-2 locus, are also good rejectors of most bone marrow allografts. (89)Sr, a bone-seeking isotope, irradiates marrow but not other lymphoid organs and abrogates resistance to marrow allografts without suppressing T- or B-cell functions. Thus, marrow-dependent effector cells (M cells) seem to resist allogeneic stem cells. To test if the genetic resistance to Friend virus (FV) is also mediated by M cells, B6 mice were treated with (89)Sr using a dosage schedule known to abrogate resistance to allogeneic marrow cells. 9 days after FV infection of such mice, the spleens showed malignant erythroblastosis which could not be suppressed by prior hypertransfusion, a procedure which suppresses physiologic erythropoiesis. Such (89)Sr-treated B6 mice also supported extensive virus replication, while control mice did not. FV markedly suppressed the ability of (89)Sr-treated B6 mice to produce antisheep red blood cell (SRBC) antibodies, a feature seen normally only in genetically susceptible mice. Thus, (89)Sr-treated B6 mice behaved in these respects as if they were susceptible to FV. When increasing doses of (89)Sr were administered to B6 mice, a dose-related loss of resistance to FV was seen. Therefore, it appears that (89)Sr-sensitive M cells mediate the genetic resistance to FV. The results of experiments with (89)Sr indicated that genetically resistant mice would be expected to possess target cells which are susceptible to transformation by FV. To verify this corollary, bone marrow cells from B10.D2 (Fv-2(rr)) mice were transplanted into previously infected and lethally irradiated DBA/2 (Fv-2(ss)) recipients which share the same H-2(d) alleles. 5-15 days later, the spleens of DBA/2 primary recipients yielded transformed cells which were capable of producing splenic tumor colonies upon transplantation into adult, unirradiated B10.D2 secondary recipients. Various control experiments clearly indicated that the tumor colonies so induced were of B10.D2 marrow origin. This indicated that B10.D2 stem cells could be transformed when allowed to interact with FV in the spleens of susceptible DBA/2 mice. However, 30 days after transplantation of B10.D2 bone marrow cells into DBA/2 recipients, no transformed cells were detected. Apparently, in the 30-day interval precursors in the B10.D2 marrow gave rise to mature M cells which resisted the leukemic process. Since M cells recognize hybrid or hemopoietic histocompatability antigens expressed on primitive normal and transformed hematopoietic cells, we suggest that M cells may exert surveillance by rejecting leukemic cells. Thus, marrow transplantation from genetically resistant donors may provide a new mode of treatment for leukemia, by providing precursors of M cells and other immunocompetent cell types.

Inheritance of susceptibility to Friend mouse leukemia virus. 8. Effect of a single genetic locus on the pathogenesis of the leukemia.

AbstractThe effect of the Fv locus on the pathogenesis of Friend virus‐induced leukemia was studied with two pairs of mouse strains which are congenic except for the locus. The rapid spleen enlargement or spleen focus formation which is characteristic of Friend virus infection occurred in the mice with Fvs/Fvs genotype, DDD and C57BL/ 6‐Fvs. These early responses were not seen in the mice with Fvr/Fvr genotype, DDD‐Fvr and C57BL/6. When the infected Fvr/Fvr mice were observed for 150 days, all of the DDD‐Fvr mice, but none of the C57BL/6, developed splenomegaly. Histological studies of the DDD‐Fvr mice revealed erythroblastic, lymphatic or granulocytic leukemia, or myeloproliferative disorder with a prominent megakaryocyte proliferation. This observation indicates that Friend leukemia virus is able to induce various types of leukemias in mice and the early characteristic responses are completely abrogated in the mice with Fvr/Fvr genotype. Involvement of genetic factor (s) other than the Fv locus in the late‐occurring leukemias was suggested.

Effect of friend leukemia virus infection upon polycythemia and δ-aminolevulinic acid synthetase activity in murine spleen and liver

Effect of friend leukemia virus infection upon polycythemia and δ-aminolevulinic acid synthetase activity in murine spleen and liver

Porphobilinogen and porphyrin synthesis in splenomegaly of some murine leukemias.

SummaryThe production of porphobilinogen and porphyrins by spleen extracts of Friend virus (FV) infected mice depends on the initial enzymic activity in the organ and varies with the amount of virus injected and the strain of mice employed. The increase in the enzymic activity appears to be as sensitive a measure of FV infection as was the splenomegalic response. Only minor changes in the enzymic activity were observed during the development of splenomegaly induced by leukemia L1210.

Interference in Friend virus infection in vitro.

Interference in Friend virus infection in vitro.

Plasma lactate dehydrogenase and spleen heme biosynthetic activity following Friend and Rauscher leukemia virus infections

The progression of plasma lactate dehydrogenase and heme biosynthetic activity in the spleen was followed in mice infected with Friend and Rauscher viruses. Plasma lactate dehydrogenase increased progressively in both viral diseases and paralleled the increase in spleen weight. Despite the similarity of the spleen weight responses, Friend virus promoted an earlier and more potent erythroid response as measured by porphobilinogen and porphyrin production than did Rauscher virus. The possible origin of the substantial increases in plasma lactate dehydrogenase is discussed.