Mouse Leukemia Virus Research Articles

Replication of murine leukemia virus in heterologous cells: Interaction between ecotropic and xenotropic viruses

Studies reported here demonstrated that phenotypic mixing readily occurred between an endogenous xenotropic type C virus (TB-MuX) induced from TB cells of CFW/D mouse and ecotropic Moloney murine leukemia virus (Mo-MuLV). The pseudotypes generated were used to investigate the level of restriction of replication of these viruses in their respective nonpermissive cells. As a pseudotype of TB-MuX, Mo-MuLV was transmitted to canine (D-17) cells which were once restrictive to Mo-MuLV infection. Pseudotypes of TB-MuX genomes with Mo-MuLV envelopes were also transmitted back to TB cells. It was found that the replication of TB-MuX in TB cells was only detected in the presence of ecotropic MuLV. Several D-17 clonal cell lines infected with temperature-sensitive (ts) mutants of Mo-MuLV were isolated. These D-17 cell lines produced only ecotropic virus and were still susceptible to superinfection by TB-MuX. Virus produced by the is mutant-infected D-17 clones had the same is phenotype and host-range properties as viruses produced by is mutant-infected TB cells. The viral titers produced by the is mutant-infected D-17 clones at the permissive temperature varied from clone to clone by 2–3 log, but over 10 or more passages the titer of a given clone only varied two- to three-fold. In addition, the high producers of is mutant-infected D-17 clones produced as much virus at the permissive temperature as is mutant-infected TB cells. It was also shown by measuring infectivity in Mouse S +L − cells that at the nonpermissive temperature the temperature-sensitive defect of ts3 was complemented by TB-MuX. In contrast, using the same approach, it was found that at the nonpermissive temperature TB-MuX did not complement the temperature-sensitive defect of ts7. On subcloning D-17ts3aC1-17, a D-17 clonal line which produced relatively high titer of ecotropic virus, all producing subclones tested remained high producers like the parent clone, except one which produced about 2 log less virus than the rest of the productively infected subclones. In addition, 3 of the 32 subclones isolated were nonproducers. Radioimmunoprecipitation of intracellular virus-specific proteins of one of the nonproducer subclones, cl-19, showed the presence of gp and gag precursor proteins. On subcloning D-17ts3c1-2, a low producer ts3-infected D-17 cloned line, 93 subclones were isolated and 23% of these subclones failed to detect the presence of intracellular virus-specific proteins. The viral titer of the productively infected subclones tested did not differ significantly from that of the parent clone. The Mo-MuLV-infected D-17 clonal cell lines were used to develop an indirect assay for TB-MuX.

Genetic control of sensitivity to Moloney leukemia virus in mice. III. The three H-2 linked Rmv genes are immune response genes controlling the antiviral antibody response.

It has been shown previously that three different H-2-associated genes control the resistance to viremia and leukemia in Moloney virus-infected mice: Rmv. 1, mapping to the I-A or less probably K regions; Rmv. 2, mapping to the I-C, S or G regions and Rmv. 3, mapping to the D or T regions. Experiments have been performed to determine the role of these genes in the control of the antibody responses directed against Moloney murine leukemia virus (M. MuLV) virions and/or leukemic cells. The inoculation of infectious M.MuLV failed to provide conclusive responses due to unequal replication of the virus in different inbred strains resulting in variable antigenic stimulations and/or in vivo antibody absorptions. The use of inactivated M.MuLV as antigen allowed to avoid these problems. It showed that (2) the IgG-specific antiM.MuLV response is controlled by H-2 linked genes, (b) a clear correlation exists between high or low-responder phenotypes and the resistance or susceptibility to M.MuLV infection and (c) all three Rmv genes behave like immune response genes. These results were not surprising for Rmv. 1 and Rmv. 2 which map in the I region of the major histocompatibility complex. It was more puzzling for Rmv. 3. Further experiments are necessary to determine the exact mechanism by which this gene controls the immune response.

In vitro gamma irradiation of leukemic cells in mice, rats, and guinea pigs.

In vitro gamma irradiation of virus-induced (Gross) mouse leukemia cells at doses of 350-1600 rads (1 rad = 0.01 gray) had no effect on their ability to induce leukemia, usually within 2 weeks, after transplantation into syngeneic mice. However, when cells irradiated at doses of 2000-20,000 rads were transplanted, they induced leukemia after a latency period exceeding 2.5 months, similar to the results observed in mice inoculated with filtered mouse leukemia extracts. Similar results were also obtained after irradiation of leukemic cells derived from rats in which leukemia had been induced by rat-adapted mouse leukemia virus. Apparently, gamma irradiation at a dose of, or exceeding, 2000 rads, inhibits the ability of mouse and rat leukemic cells to induce leukemia after transplantation into syngeneic hosts; however, it does not inactivate the virus carried by such cells nor prevent it from inducing leukemia. [In previous experiments, doses of more than 4,500,000 rads were needed to inactivate the passage A (Gross) leukemia virus carried in either mouse or rat leukemic cells.] In vitro gamma irradiation of L2C guinea pig leukemic cells at doses of 750-2500 rads had no apparent effect on their ability to induce leukemia after transplantation effect on their ability to induce leukemia after transplantation into strain 2 guinea pigs. However, irradiation at doses of 3250-20,000 rads inactivated their ability to do so. The morphology of mouse, rat, and guinea pig leukemic cells and the virus particles present in such cells was not affected by irradiation at doses of 20,000 rads.

Genetic control of sensitivity to Moloney leukemia virus in mice. IV. Phenotypic heterogeneity of the leukemic mice.

Genetic control of sensitivity to Moloney leukemia virus in mice. IV. Phenotypic heterogeneity of the leukemic mice.

Genetic and nongenetic factors in expression of infectious murine leukemia viruses in mice of the DBA/2 × RF cross

Mice of the RF and DBA/2 strains possess endogenous ecotropic murine leukemia virus (E-MuLV) genomes but express only low to undetectable levels of infectious virus in their lymphoid tissues. F1 mice of this cross showed high levels of infectious E-MuLV if DBA/2 was the maternal parent but very low levels if RF was the maternal parent. E-MuLV expression, if present, was always higher in the spleen than in the thymus. Studies of reciprocal backcross generations with both parental strains indicated that the presence of the virus was governed by a single dominant autosomal locus present in the RF strain, and that RF females, but neither RF males nor DBA/2 females or males, transmitted a non-Mendelian factor which powerfully suppressed virus expression in their progeny. Some but not all (DBA/2♀ × RF♂)F1 females also possessed the capacity to transmit this maternal suppression to their progeny. Xenotropic murine leukemia virus (X-MuLV) showed a different pattern of expression in this cross. In the thymus it was detected in a minority of DBA/2 and in no RF mice; in crosses the presence of X-MuLV in this organ was independent of the presence of E-MuLV. In the spleen, X-MuLV was detected only in a percentage of E-MuLV-positive mice. The maternal factor from RF mothers which suppressed E-MuLV did not suppress thymic expression of X-MuLV. Skin painting with 3-methylcholanthrene induced a high incidence of thymic lymphoma in mice of both parental strains and in F1 hybrids, all of which normally show only low incidences of the diseases; the treatment did not induce markedly increased expression of E-MuLV or X-MuLV in mice of either parental strain, although it did abrogate the diminution of E-MuLV titers seen with age in (DBA/2♀ × RF♂)F1 mice beyond the age of three months.

Different lymphoid cell targets for transformation by replication-competent moloney and rauscher mouse leukemia viruses

Mouse leukemia viruses (MuLV) have been reported to induce tumors involving cells within the T lymphocyte lineage. In the present study, striking differences were demonstrated in the target cells for in vivo transformation by two clonal replication-competent type C viruses, Moloney- and Rauscher-MuLV. Moloney-MuLV-induced tumors and lymphoma cell lines exhibited Thy.1 antigen in the absence of detectable Fc or C3 receptors, indicating their T cell origin. Rauscher-MuLV primary tumors and lymphoma cell lines of the same mouse strain, however, invariably exhibited Fc receptors in the absence of Thy.1 antigen, suggesting that these tumors were of the B lymphoid lineage. The pattern of immunoglobulin synthesis by individual Rauscher-MuLV tumor cell lines was determined by both biosynthetic and radioimmunologic techniques. Rauscher-MuLV lymphoma lines invariably expressed immunoglobulin heavy (mu) chain in the absence of detectable light (kappa or lambda) chains. These findings establish that the target of neoplastic transformation in response to Rauscher-MuLV is an immature cell within the B lymphoid lineage. The demonstration of different target cells for transformation by well characterized clonal strains of mouse leukemia virus should aid in elucidating the mechanisms by which these viruses induce malignancy.

Effect of interferon on mouse leukaemia virus (MuLV). V. Abnormal proteins in virions of Rauscher MuLV produced in the presence of interferon.

Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of non-infectious particles ('interferon' virions) containing the structural proteins coded by the env and gag genes as well as additional virus polypeptides. The major glycoprotein detected in the control virions is gp71, but 'interferon' virions contain in addition an 85K mol. wt. (gp85) glucosamine-containing, fucose-deficient glycoprotein. This is recognized by antiserum to MuLV and may be related to env pr85. Surface iodination of intact virions indicates that gp71 and gp85 are the two major components of the external envelope. However, whereas in control virions gp71 associates with p15E (gp90), this complex was not detected in 'interferon' virions. Analysis of radio-labelled (3H-amino acids or iodinated) proteins from disrupted 'interferon' virions revealed the presence of 65K, 55K, 40K, 20K and 12K mol. wt. polypeptides which could be precipitated with antiserum against MuLV. There was a distinct difference in the patterns of incorporation of pulse-labelled 3H-amino acid polypeptides into virions in the presence and absence of interferon. Those polypeptides labelled in the presence of interferon and recovered in the extracellular virions in a chase with interferon appeared to have substantially fewer copies of p30 and more of gag pr55 polypeptide than the controls. These results indicate that in the presence of interferon there are changes in the proteolytic cleavage associated with virion assembly.

Fv-2 locus controls the proportion of erythropoietic progenitor cells (BFU-E) synthesizing DNA in normal mice

We have found that Fv-2 on chromosome 9 of the mouse, the locus originally identified as a major determinant of host susceptibility ( Fv-2 s ) or resistance ( Fv-2 r ) to Friend leukemia virus in mice, also functions in uninfected animals, where it determines whether a high ( Fv-2 s ) or low ( Fv-2 r ) proportion of erythropoietic progenitor cells BFU-E are normally engaged in DNA synthesis. Adult mice belonging to five inbred strains of genotype Fv-2 rr , two inbred strains and three congenic strains of genotype Fv-2 ss , two kinds of F1 hybrid of genotype Fv-2 rs , and appropriate controls were given high specific activity 3H-thymidine intravenously for 1 hr and their bone marrow and spleen cells were assayed for surviving BFU-E at 7 days and CFU-E at 2 days in plasma culture. A high proportion of BFU-E in all Fv-2 ss and Fv-2 rs mice were killed, but all or nearly all of the BFU-E in Fv-2 rr mice survived exposure to 3H-thymidine. The allelic difference at Fv-2 had no significant effect on the proportion of other progenitor or stem cells (CFU-E, CFU-C or CFU-S) normally undergoing DNA synthesis in the hemopoietic tissues, or on the hemoglobin concentration, red blood cells, hematocrit, total or differential white blood cell counts in the peripheral blood of these animals. While a few or no BFU-E were killed by 3H-thymidine in adult B6 ( Fv-2 rr ) mice, a high proportion of BFU-E were killed by 3H-thymidine in these animals when they were less than 7 weeks old. Bleeding of adult B6 mice or lethal irradiation followed by repopulation by syngeneic bone marrow cells rendered a high proportion of their BFU-E vulnerable to 3H-thymidine. BFU-E of adult B6 mice which in vivo were unaffected by 3H-thymidine were rapidly killed when exposed to 3H-thymidine in vitro. Fv-2 thus seems to act at or near the G 1-S or G 0-S boundary to influence the rate or probability of transition between the nonDNA-synthesizing and the DNA-synthesizing states of BFUE. The gene that controls susceptibility or resistance to a murine erythroleukemia virus appears also to be a regulatory gene that controls the proliferative behaviour of normal cells at a specific stage of erythropoietic differentiation.

Nucleotide sequence of a cloned murine leukemia virus DNA fragment.

The oncogenic retroviruses can be divided into three classes. One class, exemplified by avian Rous sarcoma virus (RSV), is replication-competent and carries a transforming gene, in this case the src gene. The second class carries a transforming gene, but is replication-defective and therefore requires a helper virus for growth. Examples of these viruses are Abelson, Moloney sarcoma, and spleen focus-forming viruses. The third, and to us most intriguing, class consists of those viruses that are replication-competent, but do not carry extra transforming genes. Mouse leukemia viruses, those designated mink-cell focus-inducing (MCF) and discussed in detail by Rowe et al. (this volume), are of this sort.

Genetic Control of Sensitivity to Moloney Leukemia Virus in Mice

Abstract The level of viremia and the appearance of leukemias were studied after inoculation with Moloney leukemia virus (M-MuLV) in different H-2 congenic strains of mice. The viremia was regularly measured on individual mice with a radioimmunoassay of the major internal virion component p30. Three genes within the major histocom-patibility complex controlled the level of circulating virus. Two of them, called Rmv.1 and Rmv.2, appear to be located in the I region, respectively, in the IA, and the IC-S or G regions. The third gene, Rmv.3, was mapped to the D end of the complex in the D or T region. Crosses between resistant and sensitive strains demonstrated that the H-2 associated resistance was inherited as a dominant or semi-dominant Mendelian trait. Rmv.1, Rmv.2, and Rmv.3 were shown to complement for resistance in trans when the hybrids between sensitive strains were examined. A good correlation was found between viremia and the appearance of leukemias, the most viremic strains being also the most leukemic. Nevertheless, additional non-H-2 genes must control viremia and/or the appearance of leukemia since, despite high levels of viremia, some sensitive strains do not become leukemic.

Carcinogenic chemicals enhance mouse leukemia virus infection in contact-inhibited culture: A new simple method of screening carcinogens

Carcinogenic chemicals enhanced infection in contact-inhibited cells with mouse leukemia virus. A correlation was found between the enhancing effects and the carcinogenicities of the 40 chemicals tested. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate did not enhance viral infection.

Attempt to immunize guinea pigs against L2C leukemia with leukemia cells inactivated by gamma irradiation.

In previous studies, intradermal inoculation of small doses of L2C leukemic cell suspensions into strain 2 guinea pigs induced immunity against challenging reinoculation with leukemic cells; however, 50% of the guinea pigs developed leukemia in the course of immunization. We have now attempted to induce immunity by inoculation of L2C leukemic cells inactivated by in vitro gamma irradiation ranging from 750 to 8000 rads (1 rad = 0.01 gray). In preliminary experiments, irradiation with 750 to 2750 rads had no significant effect on leukemogenic potency of leukemic cells; however, doses exceeding 3000 rads inactivated the leukemogenic potency of L2C cells. Eighty-nine guinea pigs that survived intradermal, subcutaneous, or intraperitoneal inoculations with irradiated (1000-8000 rads) L2C cells were subsequently challenged by reinoculation with nonirradiated leukemic cells, and 83 of them (93%) developed leukemia. L2C leukemic cells contain spherical particles, about 103 nm in diameter; it is reasonable to assume that these particles represent the causative virus responsible for the development of L2C leukemia. Inactivation of leukemogenic potency of L2C leukemic cells by gamma irradiation in vitro does not necessarily imply that the virus particles consistently present in these cells were also inactivated. In previous experiments carried out on mouse leukemia, doses exceeding 1,000,000 rads were needed in order to inactivate the mouse leukemia virus in vitro.

Protective effect of immunization with nonviral antigens against Friend leukemia virus in mice

DBA/2 mice immunized with poly(A).poly(U) complexed with methylated bovine serum albumin and emulsified in Freund's complete adjuvant were protected against challenge with Friend leukemia virus. There was no correlation between the level of antibody to the immunogen in the prechallenge serum and induced resistance to the virus. Although prechallenge sera of mice given the same amount of the duplex in a single inoculum bound 9.7% of poly(A).poly([(3)H]U) input, as compared to 45.3% bound by the prechallenge sera of mice given the immunogen in divided doses, both groups of mice were equally resistant to infection. Immunization with two other nonviral agents, bovine serum albumin fraction V or dinitrophenylated keyhole limpet hemocyanin, induced the same level of protection. A sparing effect of approximately 10(1.5) in infectivity was afforded the immunized mice. Immunization with either poly(A).poly(U) alone or with the carrier methylated bovine serum albumin was ineffective. In addition to antibodies to the respective immunogens, the prechallenge sera of the immunized mice also contained antibody to Friend leukemia virus gp71. The presence of such viral antibodies was not always related to resistance to infection by Friend virus. Some immunized mice that survived infection did not have gp71 antibody in their serum before challenge, and mice immunized with poly(A).poly(U) alone were susceptible to infection, although their prechallenge sera contained antibody to gp71. The mechanism involved in the induction of resistance to infection is not known. The effect may be mediated through a modification of the expression of both endogenous and exogenous type C viruses and affect immunological mechanisms controlling cellular responses.

Antigenic Properties and Molecular Weights of Murine Leukemia Virus-Binding Proteins

A murine T lymphoma cell line, WEHI-22, has been studied for the presence of murine leukemia virus-binding proteins and for the presence of cell surface molecules that share antigens with mouse immunoglobulins. With surface radioiodination, detergent disruption, and immunoprecipitation, a 60 to 70,000-dalton molecule has been described that is recognized by chicken anti-mouse immunoglobulin serum. In competition experiments this molecule cross-reacts with highly purified mouse IgM myeloma proteins. A cell surface molecule of similar size can be shown to bind to mouse leukemia viruses. Pre-precipitation of the WEHI-22 cell surface material with chicken anti-mouse immunoglobulin removes the material binding to leukemia viruses.

Amino Acid Sequence Homology Between Histone H5 and Murine Leukemia Virus Phosphoprotein p12

The amino terminal acid sequences of several mouse leukemia virus phosphoproteins (p12) show definite homology with the amino terminal conserved region of H5 histones, the phosphorylated nuclear proteins of nucleated erythrocytes. Differences in the amino acid compositions of the two groups of proteins seem to rule out the possibility that they evolved from a single common ancestral gene. The finding of sequence homology between viral p12's and cellular histones, however, is consistent with evolution of retrovirus structural proteins by a process of differentiation from preexisting cellular genes. The conserved primary and secondary structure at the amino terminal region, common to both groups of proteins, may be related to their common function of nucleic acid binding modulated by phosphorylation.

Properties of mouse leukemia viruses XVI. Suppression of spontaneous fatal leukemias in AKR mice by treatment with broadly reacting antibody against the viral glycoprotein gp 71

AKR mice were treated early in life with antibodies prepared in a goat against the major glycoprotein gp71 of Friend murine leukemia virus (FL V) and evaluated over a period of up to 2 years for various parameters associated with AKR thymoma/lymphoma. If both the mothers and offspring were given the antibody, suppression of AKR disease was observed to the degree that the 50% incidence of leukemia was delayed by about 1 year. A lesser effect was found if the treatment was initiated at an age of 3 days and application of the antibody to either mothers alone, or treatment of the mice at later times (39 days) was not successful. Thus, it was concluded that the critical period for treatment is between birth and the first few days of life. Postmortem examination of animals that were successfully treated during this early period revealed a significant proportion exhibiting a unique leukemic pattern. In addition, several died from nonleukemic neoplasms as well as other causes. Levels of virus or antiviral antibodies were also determined at various times in individual mice which had been segregated in family groups. The results demonstrated that overall, successful antibody treatment resulted in (1) suppression of virus and (2) development of significant levels of antiviral antibodies. In contrast, control mice exhibited the opposite pattern: high levels of virus and no detectable antibody. Moreover, it could be shown that antibody treatment reduced the incidence of MCF-like recombinant virus isolation. A hypothesis is presented which suggests that the main function of the administered antibody is to disturb a key event occurring during the early stages of life of the AKR mouse, which is of critical importance for the development of leukemia from 6 months of age onwards.

35] Purification and assay of murine leukemia viruses

This chapter describes the techniques for growing murine leukemia viruses in tissue culture and discusses methods for purifying virus in sufficient quantities for subsequent biochemical studies. As the success of these techniques involves determinations of viral titer, several of the commonly used, quantitative in vitro assays for viral replication are also described. Murine type C viruses have been classified into two major groups based on their potential for producing neoplastic disease. The mouse leukemia viruses (MuLVs) can replicate in cultures of fibroblastic and epithelioid cells and generally do not produce cytopathic effects or morphological transformation. Mouse sarcoma viruses (MSVs) are defective in their ability to replicate, require “helper” MuLVs for their continued propagation in cell culture, and produce morphological transformation of susceptible cells in vitro.

T-cell lymphoma induction by radiation leukemia virus in athymic nude mice.

We report the development of extrathymic lymphoblastic lymphomas in RadLV-inoculated congenitally athymic nude mice. Thus, a leukemogenic virus which appears to require the presence of a thymus for its replication in normothymic mice can infect and transform target cells in the absence of this organ in the athymic host. The cells of one of these lymphomas have been established in vitro as a permanent cell line, BALB/Nu1. This cell line as well as a lymphoma induced in NIH/Swiss nude mice exhibit several T-cell markers, including terminal deoxynucleotidyl transferase activity, Thy-1.2, and Ly-2.2, but not Ly-1.2 nor TL. Ig determinants were not detected. The characteristics of the tumor cells support the view that cells with T-cell markers may normally exist in nude mice and undergo neoplastic transformation and clonal expansion after infection with a leukemogenic virus. The alternative possibility that virus-induced differentiation of prothymocytes may lead to the expression of Thy-1.2 and Ly-2.2 antigens is also considered. BALB/Nu1 cells release large numbers of type C viral particles. The virus, designated radiation leukemia virus (RadLV)/Nu1, has RTase activity and the protein profile characteristic of murine leukemia virus (MuLV). In radioimmunoassays, it cross-reacts completely with RadLV/VL3, a virus obtained from RadLV-induced C57BL/Ka thymic lymphoma cells in culture, and slightly with a xenotropic virus (BALB:virus-2) and with AKR MuLV. On inoculation into C57BL/Ka mice it has thymotropic and leukemogenic activity. In vitro it is B-tropic, poorly fibrotropic, and has limited xenotropic activity. Thus, RadLV/Nu1 appears to be biologically and serologically similar or identical to its parent virus, RadLV.

Immunofluorescent analysis of murine leukemia virus-infected cells by flow microfluorometry

Flow microfluorometric assay with a Cytofluorograf model 4801 in combination with immunofluorescence was applied to the quantitative assay of cells exogenously infected with mouse leukemia viruses and to the chemical induction of virus in AKR cells. The Cytofluorograf provides sensitivity equal to the visual method and is capable of assaying up to 5000 cells/sec with specificity equivalent to that of the direct visual immunofluorescence assay, thereby providing a large, statistically valid sampling in a fraction of the time required by visual counting. Flow microfluorometry also provides a method of quantitatively resolving fluorescent cell populations on the basis of relative size and degree of fluorescence.

Relative loss of oncogenic potency of mouse leukemia virus (Gross) after prolonged propagation in tissue culture.

Since the initial development of the "passage A" mouse leukemia virus in 1957, this virus has been propagated in our laboratory by serial passage in newborn C3H(f) mice. At the present time, 10(-2)-10(-3) dilutions in physiological saline solution of this mouse-passaged virus induce lymphatic leukemia in practically all inoculated mice after a latency of 3-5 months. On the other hand, when the same virus was propagated on NIH 3T3 mouse embryo cells in tissue culture for more than 10 years, its leukemogenic potency became considerably reduced. Recent bioassay experiments carried out in our laboratory demonstrated that after such prolonged propagation in tissue culture this virus now induced leukemia in less than 15% of the inoculated suckling C3H(f) mice; only undiluted or 10% dilutions of the tissue culture fluid (very occasionally 10(-2) or 10(-3) dilutions) induced leukemia after a prolonged latency varying from 5.5 to 18 months. The passaged and the tissue-culture-grown virus strains are identical immunologically and indistinguishable in their morphology when examined by electron microscopy. The tissue-culture-grown virus, attenuated in its leukemogenic potency, does not, however, confer immunity against a challenge with the mouse-passaged virus.