Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis.

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.

Requirement of the CD8+ or CD4+ T lymphocyte subsets for the rejection of lymphoma and fibrosarcoma grafts studied in gene knockout hosts.

Rejections of the retrovirus induced lymphomas (ALC and RBL-5) and the methylcholanthrene (MCA) induced fibrosarcoma (MC57X) grafts were tested in syngeneic CD8 and CD4 single and double knockout C57BL/6 mice. The results with the lymphomas showed that the CD8+ T cell deficiency prevented the development of rejection response induced by immunization. Deficiency of the CD4+ T subset abrogated also the rejection of ALC. Immunity against the fibrosarcoma cells developed in both type of single knockout mice, but not in the ones which lacked both CD4+ and CD8+ T cells. Thus CD8+ T cells were required for rejection of the lymphoma cells, while the CD4+ T cells only mediated a weak response. In absence of CD8+ T cells, CD4+ T cells were sufficient to reject the fibrosarcoma cells.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

[Submicroscopic features of cells in the microenvironment of hematopoietic development of virus-induced Rauscher leukemia]. / Submikroskopicheskie osobennosti kletok mikrookruzheniia krovetvornykh organov v protsesse razvitiia virusindutsirovannogo leikoza Raushera.

The study was made of submicroscopic changes in the cells of bone marrow and splenic microenvironment in mice developing virus-induced Rauscher leukemia. As shown by electron microscopy, ultrastructural cytochemistry and immunocytochemistry, ultrastructure of the complexes from the stromal and hemopoietic cells underwent noticeable alterations as early as the first days after the virus introduction. This suggests that bone marrow is the primary target of the virus in Rauscher leukemia. Affections of the macrophages, dendrite, interdigital and lymphoid cells of the spleen reflect their participation in the body defenses against the virus. Progressive shift of erythropoiesis from the bone marrow into the spleen is related to morphofunctional changes in the microenvironmental cells. The findings may be useful in consideration of cellular pathogenetic aspects of acute leukemia.

The role of CD4 and CD8 T cells in the graft-versus-leukemia response in Rauscher murine leukemia.

Using a mouse model for MHC-matched unrelated donor transplantation, the relative influences of the CD4 and CD8 T cell subtypes on graft-versus-leukemia (GVL) were examined in a murine erythroleukemia induced in SJL/J mice by the injection of Rauscher virus. Following leukemia induction, the mice were given 9.5 Gy of total body irradiation (TBI) and injected with mixed marrow and spleen cells from normal MHC-matched--but minor histocompatibility mismatched--B10.S donors. Prior to their injection these donor cells were selectively depleted ex vivo for either CD4, CD8 or Thy-1 by exposure to the appropriate monoclonal antibody (MoAb) plus complement. Following transplant the recipients were observed for 20 weeks, along with parallel control groups, for survival, leukemia relapse, graft failure and graft-versus-host disease; 98% of the controls receiving no transplantation therapy died of leukemia. Among the controls that received TBI plus undepleted B10.S cells 30.9% died of leukemia relapse, but another 34.2% survived free of any clinical evidence of their leukemia. Donor cell depletion for Thy-1 increased the relapse to 68.8%, while survival fell to 10.4%. CD8 depletion resulted in a relapse of 55.6%, with a survival of 19.4%. By contrast, CD4 depletion had no effect on relapse, but did significantly increase the incidence of graft failure. At the end of the 20 weeks additional tests were run to determine whether those transplant survivors that had remained leukemia-free were also free of any residual Rauscher virus. Those tests showed that they were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of mouse retroviral disease by bioactive glutaminase-asparaginase.

Glutamine depletion strongly inhibits the replication of Rauscher murine leukaemia retrovirus (RLV) in vitro. Pseudomonas 7A glutaminase-asparaginase (PGA), capable of depleting glutamine and asparagine for prolonged periods, was used to determine the therapeutic effectiveness of glutamine depletion in mice infected with RLV or Friend virus. During PGA treatment of viraemic animals, serum reverse transcriptase activity fell to control levels and infected animals did not develop splenomegaly. The therapeutic results obtained with PGA compared favourably with those of azidothymidine given intraperitoneally at 30 mg/kg/day. Western blots performed on splenic tissue from control and treated animals indicated that glutamine depletion prevented readthrough of an amber codon at the gag-pol junction, stopping translation of viral mRNA at that point. Treatment of RLV-infected animals with PGA resulted in nearly a 200% increase in mean survival time even when therapy was initiated late in the course of the disease. To our knowledge, this is the first demonstration that a nutrient required for viral replication can be enzymically depleted in vivo to inhibit viral replication.

Castanospermine vs. its 6-O-butanoyl analog: a comparison of toxicity and antiviral activity in vitro and in vivo.

Inhibitors of glycoprotein processing, such as castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), have been shown previously to inhibit human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured human cells. In prior experiments, we have tested the toxicity and antiviral efficacy of castanospermine in mice infected with the Rauscher murine leukemia virus (RLV). When compared with 3'-azido-3'-deoxythymidine (AZT, zidovudine), castanospermine was less effective and more toxic. Since the 6-O-butanoyl analog of castanospermine was previously found to have a more favorable activity profile than the parent compound against HIV-1 in cultured cells, we compared the antiviral efficacy of both compounds in parallel in vitro and in vivo in the RLV system. Plaque formation in the XC assay was inhibited with a 50% inhibitory concentration (IC50) of 2.4 microM for the 6-O-butanoyl analog of castanospermine, as compared to 9 microM for castanospermine. For both compounds, concentrations resulting in significant cytotoxicity were about ten times higher. Both compounds significantly decreased HIV-1 env-induced syncytium formation in a novel in vitro assay. In RLV-exposed mice, the 6-O-butanoyl analog showed no advantage over the parent compound: both curves for toxicity as well as antiviral efficacy were super-imposable. We conclude that the 6-O-butanoyl analog of castanospermine as well as castanospermine itself are active antiviral agents in mice and that prolonged oral administration is tolerable. However, in comparison to AZT, their antiviral activity profiles are less favorable.

N-carboxymethylchitosan-N,O-sulfate as an anti-HIV-1 agent.

N-carboxymethylchitosan-N-O-sulfate (NCMCS), a sulfated polysaccharide derivative of chitin, inhibited the propagation of the human immunodeficiency virus type 1 (HIV-1) in human CD4+ cells and that of Rauscher murine leukemia virus (RLV) in murine fibroblasts. A dose-dependent inhibition of both viruses was observed without significant cytotoxicity. NCMCS blocked the binding of HIV-1 to human CD4+ target cells and competitively inhibited HIV-1 reverse transcriptase. Thus, NCMCS may prevent HIV-1 infection by inhibiting viral adsorption to the CD4 receptor and reverse transcription of the viral genome.

Cell surface receptors for ecotropic murine retroviruses: mobile membrane proteins that mediate binding and slow endocytosis of the viral envelope glycoprotein.

The gp70 envelope glycoproteins of ecotropic murine leukemia viruses bind to receptors that occur only on mouse and rat cells and on interspecies hybrid cells that contain mouse chromosome 5. A substantial fraction of the gp70 that was bound specifically by these criteria remained undegraded and accessible to extracellular labeling reagents for many hours. Accordingly, cells with ecotropic receptors could be labeled specifically. As seen by immunofluorescence microscopy, the gp70-receptor complexes were uniformly dispersed on mouse fibroblast plasma membranes. These complexes were mobile, and they aggregated into patches when crosslinked by antibodies at 37 degrees, but not when membrane lipid fluidity was frozen at 0 degrees. Ecotropic receptors still bound gp70 specifically after cells were fixed with 3.7% formaldehyde, but these receptors could not be patched, indicating that they were nondiffusible. Viable cells slowly endocytosed gp70-receptor complexes at 37 degrees (approximate half-life 5-7 hr) and the gp70 was then proteolytically degraded in lysosomes. In the presence of 20 microM chloroquine, a lysosomal inhibitor, undegraded gp70 was seen to slowly accumulate in these intracellular organelles. These results suggest that ecotropic receptors mediate a slow internalization of attached ligand. Long-lived binding of gp70 onto surfaces of uninfected cells may explain important features of viral-induced leukemia, the host immune response, and immunosuppression.

In vivo analysis of castanospermine, a candidate antiretroviral agent.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine), an inhibitor of glycoprotein processing, has been shown to inhibit the human immunodeficiency virus type 1 (HIV-1) with acceptable toxicity in cultured cells. In contrast to reverse transcriptase inhibitors, castanospermine targets host enzymes. We have analyzed castanospermine in murine systems, using cultured cells as well as live animals. Plaque formation by Rauscher murine leukemia virus (RLV) was inhibited with a median inhibitory concentration (IC50) of 2 micrograms/ml. RLV-exposed BALB/c mice treated with a 20 day course of castanospermine starting 4 h postinoculation showed a dose-dependent inhibition of splenomegaly. Oral castanospermine therapy given to chronically RLV-infected mice prolonged median survival from 36 to 94 days when compared to untreated controls (p = 0.007). Castanospermine was better tolerated orally than intraperitoneally at the same dose. Toxic effects included weight loss, lethargy, and dose-dependent thrombocytopenia. At the highest intraperitoneal dose, lymphoid depletion occurred in thymus, spleen, and lymph nodes. We conclude that castanospermine is an active antiviral agent in animals and that prolonged oral administration is tolerable; however, when compared to 3'-azido-3'-deoxythymidine in the same murine system, castanospermine was less active and more toxic.

Modulation of fibroblast hemopoietic regulatory activities by infection with cloned Rauscher helper leukemia virus.

We tested the ability of cellularly cloned Rauscher helper leukemia virus to modulate the release of hemopoietic regulatory activities by skin fibroblasts in culture. The results demonstrate that release of colony stimulating activity for granulocyte/macrophage progenitors by fibroblasts derived from BALB/c, NIH/RIV, 129/J, and DBA/2 mice was increased by virus infection. In contrast, virus infection severely impaired the ability of fibroblasts to support in-vitro granulopoiesis.

Differential expression of cellular genes in Rauscher leukaemia.

The methods of hybridization in solution and blot hybridization showed that spleen cells from BALB/c mice contain "silent" genes which can amplify and change their structure after infection by Rauscher leukaemia virus. The "silent" gene product is nuclear 35S RNA detectable by comparative electrophoretic analysis of the heterogeneous nuclear RNA from leukaemic and normal cells. About 7% of this 35S RNA is represented by the virus-specific sequences, but a major part is represented by the cellular sequences. In order to study the expression of the sequences homologous to 35S RNA in leukaemic and normal cells, hybridization in solution was used. Expression of the complete copies of 35S RNA was observed in nuclei of virus-infected cells, whereas this RNA in the cytoplasm is represented by the incomplete copies. The expression of the sequences homologous to this 35S RNA in normal mouse spleen cells was not revealed.

Specific sequences of the env gene determine the host range of two XC-negative viruses of the Rauscher virus complex.

Two viruses which do not give rise to XC plaques in the standard XC assay (XC-negative) have been isolated from the Rauscher virus (RV) complex. These viruses differ in their host range. One, R-MCF-1, is dualtropic and will therefore infect both murine and non-murine cells. However, unlike other mink cell focus-inducing (MCF) viruses, it cannot infect NIH 3T3 cells. The other, R-XC-, is ecotropic. It will infect murine cells, including NIH 3T3 cells, but does not infect mink lung cells. Analysis of hybrid viruses, in which homologous regions of the genomes of R-MCF-1 and R-XC- virus were exchanged, indicated that the NH2-terminal portion of the gp70 is responsible for the particular host ranges of these viruses. The nucleotide sequence of the env gene of R-XC- virus was therefore determined and compared with the known env sequences of ecotropic MLVs and dualtropic MCF viruses of the Rauscher and Friend virus complexes. R-XC- virus was found to be a recombinant virus. Its env gene contained sequences derived from an endogenous env gene which were closely related to those of the MCF viruses but differed from any previously described sequences. The particular properties of R-MCF-1 and R-XC- virus suggest that the two viruses arose by recombination between R-MLV and two endogenous env sequences which differ from those of the known MCF viruses. If so, this suggests that the mouse genome contains at least five env sequences which can give rise to MCF-like viruses. In addition, since the host range and interference properties of R-XC- virus are very similar to those of the previously described ecotropic recombinant viruses, it may be that the ecotropic recombinant viruses arose by recombination with the same endogenous env sequences as did R-XC- virus.

Cytoskeleton-associated Pr65gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells.

Certain temperature-sensitive (ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65gag, at 39 degrees, the nonpermissive temperature. At 39 degrees, virions released from cells infected with the various ts mutants also contained elevated levels of Pr65gag relative to virions released at 33 degrees, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr65gag accumulated in the cytoskeleton-enriched fraction at 39 degrees but not at 33 degrees. During steady-state labeling, as much as half of the intracellular Pr65gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39 degrees. At permissive temperature only 10-15% of the intracellular Pr65gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65gag in the cytoskeleton-enriched cell fraction during a short pulse at 39 degrees, but Pr65gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33 degrees. Based upon these and previous results (Edbauer and Naso, 1983), models for retrovirus assembly are described in which the association of Pr65gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.

Analysis of the interaction between Rauscher murine leukemia virus and murine cell membrane receptor by in vitro binding assay.

Binding of 125I-labeled gp70 of Rauscher murine leukemia virus (R-MuLV) by 3 murine cell lines, BALB/c-3T3, NIH/3T3 and KA-31 (Kirsten murine sarcoma virus transformed clone A-31 of BALB/c-3T3) cells was measured. The binding was a saturable process, dependent on the concentration of gp70 and on the number of cells. In no experiment could we demonstrate any quantitative utilization of gp70 in the medium. However, gp70 remaining in the spent medium could be bound to fresh cells in a subsequent incubation. BALB/c-3T3, NIH/3T3 and KA-31 cells showed similar association constants (1.2-2.5 x 10(8) M-1) for the binding. Moreover, all 3 cell lines had similar number of receptors (7.4-8.9 x 10(5)) per cell. Neither N- and B-tropism of the cells nor transformation by a sarcoma virus altered the number and type of the cell surface receptors.

Rauscher murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Rauscher murine leukemia virus was molecularly cloned in a bacteriophage Charon 4A vector after the proviral sequences were enriched by sequential RPC-5 column chromatography and sucrose gradient centrifugation. A recombinant DNA clone, lambda-RV-1, possessing a 12-kilobase-pair EcoRI insert, was shown to contain the entire 8.8-kilobase-pair leukemia virus genome flanked by rat cellular sequences at the 5' and 3' ends. This DNA fragment was biologically active, inducing the release of virion-associated reverse transcriptase activity with as little as 10 ng of DNA insert. The virus induced XC plaque formation at high titers on NIH/3T3 and BALB/3T3 cells and demonstrated identity with the parental virus in radioimmunoassays for the highly type-specific gag gene-coded p12 protein. The molecularly cloned Rauscher murine leukemia virus should be useful in studying the molecular mechanisms involved in the transformation of specific lymphoid target cells by chronic mouse leukemia viruses.

Rauscher spleen focus-forming virus: biological properties and relationship to helper viruses.

A Rauscher virus (RV)-transformed erythroid cell line, RA-1, was shown to be a non-producer cell line. RA-1 cells express not only gp51-54 env-related glycoprotein, but also gp70, which is more closely related to gp51-54 coded by a recombinant env gene than to the MuLV-R gp70. RA-1 cells could be infected by Friend, Moloney and Gross viruses, but not by the homologous Rauscher murine leukaemia virus. Rescue of spleen focus-forming activity was obtained on infection of these cells with MuLV-F or MuLV-Mol, but not with MuLV-Gross. The RNA of the RV complex resembles closely that of Friend virus (FV). It contains a 32S, presumably defective, genome, which most likely is responsible for spleen focus formation, and a 35S helper virus genome. Oligonucleotide fingerprint data suggest that RV has evolved independently of FV. Erythroid early BFU-E cells of mice infected with RV of Friend helper virus-infected RA-1 cells were shown to require no addition of conditioned medium to form large erythroid colonies (BFU-E) in the presence of only small amounts of erythropoietin.