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.

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.

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.

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.

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.

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.

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)

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.

[Mechanisms of the stimulating action of the Rauscher leukemia virus on togavirus reproduction in the bodies of BALB/c strain mice]. / Izuchenie mekhanizmov stimuliruiushchego deistviia virusa leikoza Raushera na reproduktsiiu togavirusov v organizme myshei linii BALB/c.

Reciprocal stimulation or inhibition are observed in double infection of mice with Rauscher leukemia virus (RLV) and togaviruses Sindbis or West Nile (WN). Stimulation of togavirus infections is manifested by the enhancement of the visceral phase of pathogenesis without subsequent activation of togarivus reproduction in the CNS. This effect is accompanied by enhanced togavirus replication in splenocytes, a decrease in the number of antibody-producing cells in the spleen, a decrease of antihemagglutinins titer in the blood without any significant change in the virus-neutralizing antibody and interferon titers. RLV-induced immunosuppression is termporary and of varying intensity with regard to individual parameters of immune response and to different variants of WN virus (highly virulent, poorly virulent). It is assumed that the differentiated and temporary nature of the immunosupressive effect of RLV is conducive to selective stimulation of the visceral phase of togavirus reproduction followed by inhibition of the neural phase under the influence of restored immune mechanisms and interferon. Because of the defects of humoral and cellular immunity, however, no complete elimination of togavirus occurs and conditions for its long-term persistence are created.

[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.

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.

Murine leukemia virus morphogenesis: cleavage of P70 in vitro can be accompanied by a shift from a concentrically coiled internal strand ("immature") to a collapsed ("mature") form of the virus core.

Disruption of Rauscher leukemia virus (RLV) with low levels of Nonidet P-40 yielded "immature" cores. These cores have a diameter of about 920 A, as opposed to the 1300-A diameter of RLV, possess knob-like protuberances, and contain a concentrically coiled internal strand apposed to the core shell. The two major polypeptide components of immature cores are (i) p30, the 30,000-dalton group-specific antigen, and (ii) a polypeptide that has the size and antigenic characteristics of P70, the 70,000-dalton precursor protein of the group-specific antigens of murine leukemia virus. Disruption of RLV at high ratios of Nonidet P-40 to virus yielded "mature" cores. These cores have an average diameter of 850 A, a smooth proteinaceous perimeter, and a collapsed internal strand, and they contain predominantly p30. Treatment of RLV with low levels of Nonidet P-40 for 16 hr at 22 degrees yielded cores that showed (I) a 70% decrease in the number of immature forms and concomitant increase in the number of mature forms, (II) a 60-90% decrease of P70, and (iii) a 30% increase in a 40,000- to 42,000-dalton protein. These results suggest that maturation of RLV cores is accomplished by cleavage of P70.

Characterization of clones derived from an attenuated strain of Rauscher murine leukaemia virus.

With a view to defining its subpopulations, an attenuated strain of Rauscher leukaemia virus, which comprises a majority of fragile virions, was closed by end-point dilution. The presence in the obtained clones of markers (analyzed by radioimmunoassay and isoelectric focusing) associated with Rauscher virus, together with persistent infectivity and leukaemogenicity, excluded the hypothesis that endogenous virus might have replaced the original Rauscher population. Due to the closing method employed, non fragile virions were obtained. Moreover, despite its selectivity for the lymphatic leukaemia virus component, sporadic cases of atypical erythroblastogenic leukaemia were observed.

Different lymphoid cell targets by 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.

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.

Relationship of GIX antigen expression to the glycosylation of murine leukemia virus glycoprotein.

The GIX antigen, which is expressed on the surface of thymocytes of certain mouse strains, is an antigenic determinant of the major envelope glycoprotein of murine leukemia virus (gp70). Although GIX is expressed in some mouse strains that appear to be free of virus, the antigen can also be induced in GIX- mice by infection with particular murine leukemia viruses (termed GIX+). We have investigated the envelope gene products from two closely related viruses that differ in their GIX phenotype. Analysis of the envelope protein precursors by polyacrylamide gel electrophoresis and endoglycosidase treatment indicated that the GIX+ viral protein contained six oligosaccharide chains, whereas the GIX- viral protein contained seven. The observed differences in gel electrophoretic mobilities and glycopeptide profiles of the respective glycosylated envelope gene cleavage products (gp70) may be accounted for by the presence of an additional oligosaccharide chain on the gp70 of the GIX- virus. No differences between the apparent molecular weights of the nonglycosylated product of the envelope gene (p15E) were detected. These results suggest that the GIX- virus codes for an extra glycosylation site relative to the GIX+ virus, and this oligosaccharide chain is present both on the envelope gene precursor (Prenv) and on the major cleavage product (gp70). Recent nucleotide sequence analyses of selected RNase T1 oligonucleotides from the genomes of viruses that differ in GIX phenotype have similarly suggested that there may be a correlation between the GIX- phenotype and an extra glycosylation site [Donis-Keller, H., Rommelaere, J., Ellis, R. W. & Hopkins, N. (1980) Proc. Natl. Acad. Sci. USA 77, 1642-1645]. The results of these two different approaches raise the possibility that the presence of an additional oligosaccharide chain on gp70 may, either directly or indirectly, mask the expression of the GIX antigen on the surfaces of thymocytes and virus-infected cells.

Polycythemia- and anemia-inducing erythroleukemia viruses exhibit differential erythroid transforming effects in vitro.

The transforming capabilities of FVA, RLV and FVP have been examined using an in vitro transformation assay. Treatment of bone marrow cells with FVP in vitro led to the formation of hemoglobinized erythroid bursts even when these cells were cultured in methylcellulose for 5 days without added erythropoietin (Epo). A variety of FVA and RLV preparations also produced erythroid bursts without Epo but these bursts contained significantly less hemoglobin than those induced by FVP. When very low levels of Epo were added to cultures of FVA- and RLV-infected cells, the bursts were hemoglobinized, that is, similar to FVP-induced bursts. The burst-inducing agent in FVA preparations was shown to be a virus and not Epo. Spleen focus-forming virus (SFFV) pseudotypes, derived from FVA or FVP, also produced erythroid bursts in vitro, whereas four helper murine leukemia viruses did not. These studies indicated that the SFFV component was essential for erythroid burst transformation and specified the degree of hemoglobinization in the bursts formed.

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.