Antileukemic activity of Viva-Natural, a dietary seaweed extract, on Rauscher murine leukemia in comparison with anti-HIV agents, azidothymidine, dextran sulfate and pentosan polysulfate.

An antileukemic activity of partially purified polysaccharide of an edible seaweed. Viva-Natural, against Rauscher murine retrovirus-induced erythroleukemia has been demonstrated. This antileukemic effect is compared with standard anti-human immunodeficiency virus (HIV) agents, azidothymidine (AZT), dextran sulfate and pentosan polysulfate. Pretreatment with Viva-Natural, as an immunomodulator, on day 3 prior to the virus inoculation demonstrated definite prophylactic activity, while pretreatment with the other three anti-HIV agents showed no prophylactic activity. The replication of Rauscher virus in BALB/3T3 cell cultures accompanied by direct cytopathic effect (syncytia formation) was suppressed in the presence of Viva-Natural or the other anti-HIV agents in the culture medium. In spite of the antiviral potentials of the four agents in vitro, only Viva-Natural and AZT demonstrated therapeutic efficacy against Rauscher leukemia in mice.

Coordination of cleavage of gag and env gene products of murine leukemia virus: implications regarding the mechanism of processing.

Mouse 3T6 cells infected with Murine Leukemia Virus (MuLV) were cloned to yield several sublines producing viruses distinct from one another with respect to the ratio of uncleaved to cleaved gag gene-coded polyprotein, Pr65gag. The virus produced by the cloned sublines also differed in the ratio of the env gene-coded protein, p15E, to its product, p12E. The two ratios, Pr65gag/p30 and p15E/p12E, were found to be highly correlated among the cloned cell lines. Velocity gradient separation of the virions produced by individual sublines, followed by polypeptide analysis, demonstrated that the particles were inhomogeneous with respect to extent of cleavage both of PR65gag and of p15E. The two cleavages were again highly correlated. These data indicate that the gag and env gene product cleavages are not independent events but are tightly coupled.

A rapid bioassay to monitor murine leukemia virus infection in mice using cellular gp 71 binding.

A rapid and sensitive bioassay based on the availability of cell surface receptors for the binding of purified envelope glycoprotein, gp71, of Rauscher murine leukemia virus (R-MuLV) was developed to serially monitor viral-induced leukemogenesis in individual BALB/cAnN mice. The specificity of the bioassay was demonstrated by the competition of [125I]gp71 cellular binding with murine ecotropic viruses, purified unlabelled R-MuLV envelope glycoprotein and by antiserum to R-MuLV gp71. In contrast, there was no effect on the [125I]gp71 binding level with the addition of murine xenotropic viruses, R-MuLV p30, or several other proteins. The [125I]gp71 binding level of circulating leukocytes was significantly (P less than 0.05) reduced in mice after R-MuLV infection. The reduction of cellular gp71 binding developed in two stages and the latter stage was highly dependent (P less than 0.05) on circulating infectious virus titer. Using this technique, the gp71 cellular binding levels of 48-60 individual mice can be assayed in a 4 h period. The advantages of this bioassay compared to standard immunological and tissue culture techniques used in studying retrovirus expression and viral-cell interactions are discussed.

[Inhibitory effect of remantadine hydrochloride on oncornavirus production and transformation of a continuous culture of mouse leukemia cells]. / Ingibiruiushchee deistvie solianokislogo remantadina na produktsiiu onkornavirusa i transformatsiiu perevivaemoi kul'tury leikoznykh kletok myshi.

20-methylcholantrene in a concentration of 5 micrograms/ml and exposure for 72 hours as well as in a concentration of 2.5 micrograms/ml and exposure for 72 and 240 hours caused transformation of a continuous mouse leukemia cell culture producing Rauscher leukemia virus. Remantadine hydrochloride in non-toxic concentrations (12.5 and 6.25 micrograms/ml) decreased infectious virus production by cells of this culture and prevented its transformation by 20-methylcholantrene for 6 months.

Monoclonal antibodies as immunospecific probes for virus and cell surface antigen localization with the unlabeled antibody hemocyanin bridge: a review.

Hemocyanin (Hcy) from whelks, Busycon canniculatum has been developed as a visual marker for the identification of virus and cell surface antigens by the correlative techniques of fluorescence microscopy, TEM and SEM. A series of hybridomas producing monoclonal antibodies that allow the identification of type-, group-, and class-specific antigenic determinants on the major envelope glycoprotein of mouse mammary tumor virus (MMTV) gp 52 and two group-specific monoclonal antibodies to MMTV gp 36 have been developed. We used these antibodies in the unlabeled antibody Hcy bridge for immunoelectron microscopy (IEM) and found that each gp 52 antigenic determinant was expressed on virus during all stages of morphogenesis and on the infected cell surface while the gp 36 determinants were not detected. The positive labeling of gp 52 by IEM correlated well with immuno-experiments using the 125I Staph protein A plate binding assay (125IPA). The anti-gp 36 monoclonals in contrast, however, gave positive results only in the 125IPA assay. Hybridomas to murine and primate type C virion envelope proteins [gp 70 and p15(E)] have also been developed. None of the monoclonal antibodies to murine type C virus gp 70 or p15(E) gave positive labeling by IEM when tested with Rauscher murine leukemia virus but two were positive by the 125IPA assay. Monoclonal antibodies to the gp 70 of a baboon type C virus, M-7 were positive in IEM, labeling both the cell surface and viral envelope and reacted with virus in the 125IPA assay. Since monoclonal antibodies provide a much better resolution of the molecular structure and antigenic differences of viruses, interpretations of labeling results are thoroughly discussed. Methods for testing the specificity and titer of monoclonals are presented. The unlabeled antibody Hcy bridge technique and recent advances in methodology are reviewed.

Murine leukemia virus proteins expressed on the surface of infected cells in culture.

Infection of JLS-V9 cells in culture with Rauscher murine leukemia virus induced the appearance on the cell surface of two classes of viral proteins: Rauscher murine leukemia virus gp70, and glycoproteins related to the viral core (gag) proteins with apparent molecular weights in sodium dodecyl sulfate polyacrylamide gels of 80 x 10(3) and 95 x 10(3). The latter proteins were identified by lactoperoxidase-catalyzed iodination of the cell surface and by metabolic labeling with [(3)H]mannose followed by immunoprecipitation with an antiserum directed against the major viral core protein, p30. Tryptic peptide maps of chloramine T-iodinated proteins indicated that 80 x 10(3) - and 95 x 10(3)-molecular-weight proteins were closely related. The 95 x 10(3)-molecular-weight protein from Rauscher murine leukemia virus-infected cells had a tyrosine fingerprint which was identical to that of the 95 x 10(3)-molecular-weight gag surface polyprotein of endogenous virus-producing AKR-A cells, suggesting that expression on the cell surface of glycosylated forms of gag precursor polyproteins may not be an exclusive property of leukemic thymocytes, but a more general phenomenon in murine leukemia virus infection. Tryptic fingerprint analysis of iodinated viral and cell-bound gp70's before and after desialylation indicated a lower level of glycosylation in the cell-bound gp70 population than in virions. Analysis of only surface-iodinated gp70 showed a simple pattern of exposed tryptic peptides which was very similar in Rauscher murine leukemia virus-infected cells and in AKR-A cells.

Efficient production of mammalian RNA tumor viruses in serum-free culture medium allows rapid RNA subunit purification.

A wide variety of infected mammalian cell cultures were observed to produce high levels of RNA tumor virus particles in the absence of serum for at least 12 h. Virus production was measured by yields of 50-70 S virus RNAs isolated directly from serum-free culture media by chromatography on oligo(dT)-cellulose. Yields of RNAs from viruses produced in serum-free medium were comparable to yields obtained from purified viruses produced in serum-containing medium. Subunits of viral RNAs were thermally dissociated and separated by a new sedimentation system using sucrose gradients with resolving power (in the relevant size range) equivalent to that obtained with electrophoresis in polyacrylamide gels. RNA subunits isolated directly from serum-free medium appeared to be intact as judged by poly(A) content and resedimentation. The overall approach developed here represents dramatic savings in time and effort over previous ways of producing purified RNA subunits from tumor viruses.

Early event of C-type virus budding in cells infected with a Rauscher leukemia virus temperature-sensitive mutant.

Surface replicas of ts25-infected cells reveal the organization of virus-specific knobs prior to and during the early stage of budding, and antibody-mediated ferritin labeling suggests a transmembrane association of viral envelope and core components.

Therapeutic activity of pretazettine on Rauscher leukemia: comparison with the related Amaryllidaceae alkaloids.

Comparative studies on the cytotoxicity and antileukemic activity of limited numbers of Amaryllidaceae alkaloids with pretazettine, a narcissus alkaloid, have been performed on the systems of Rauscher virus-carrier cells and the leukemic mice. Only precriwelline, a stereochemical epimer of pretazettine, has been found to be therapeutically active as that of pretazettine. The natural precursors such as haemanthamine, crinamine and 6-hydroxycrinamide were also moderately active, but the artificial final product, tazettine, was confirmed to be inert. The structure-activity relationship of pretazettine or precriwelline has been partially analyzed. Also, the predominancy of antiviral activity relative to cytotoxicity of the alkaloids has been demonstrated when compared with some standard antileukemic drugs.

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.

Glycopeptides of murine leukemia viruses. I. Comparison of two ecotropic viruses.

The glycopeptides obtained by pronase digestion of two ecotropic strains of murine leukemia virus (MuLV) were compared by gel filtration. Four different glycopeptide size classes, designated G(1), G(2), G(3), and G(4), with molecular weights of approximately 5,100, 2,900, 2,200, and 1,500, respectively, were shown to be associated with Rauscher MuLV virions grown in JLS-V9 cells. Various sugar precursors, including glucosamine, galactose, fucose, and mannose were incorporated into G(1) and G(2), suggesting that these are complex (type I) glycopeptides. The two smaller glycopeptide size classes, G(3) and G(4), were shown to be mannoserich (type II) glycopeptides. G(4) was more sensitive to digestion with endo-beta-N-acetylglucosaminidase H than G(3), suggesting that the core of G(3) may contain fewer mannose residues. Glycopeptides of the same size class as G(1) and G(2) were associated with both Rauscher MuLV and AKR-MuLV grown in III6A (mouse embryo) cells. Previous studies have shown that gp52, a proteolytic cleavage product of gp70, possessed primarily G(1) glycopeptides and that gp52 was more highly sulfated than gp70. We observed that G(1) is approximately twofold more highly sulfated than G(2), explaining the observed difference in sulfation of gp52. The unusually large size of G(1) suggested that infection with MuLV may alter the host cell glycosylation pattern. To test this possibility, glycopeptides from Sindbis virions grown in uninfected and Rauscher MuLV-infected JLS-V9 cells were compared, and no differences were observed. G(1) was not detected in Sindbis virions, indicating that acquisition of G(1) depends on properties of the virus-coded polypeptide backbone of the gp70 molecule.

Depression of Rauscher leukemia virus envelope glycoprotein gp71 binding by lymphoid cells during leukemogenesis in mice.

The availability of membrane receptors for the 71,000-dalton envelope glycoprotein (gp71) of Rauscher murine leukemia virus on splenic and thymic cells from BALB/c mice during Rauscher murine leukemia virus-induced leukemogenesis was determined utilizing a radiolabeled gp71 binding assay. Shortly after infection, the relative cellular [125I]gp71 binding level decreased, first with splenic cells (at day 7 to 10 after infection) and later with thymic cells (at day 10 to 20 after infection). The dependency of the reduction of binding on the replication of the inoculated virus was demonstrated by regression analyses using cellular gp71 binding level as the dependent variable and infectious virus titer, as well as viral gp71 and p30 levels, of spleens and thymuses from infected mice as independent variables. With each independent variable, the reduction of gp71 binding for both cell types was highly dependent (P less than 0.01) on the level of virus detected in their respective organ. In the early stages of leukemogenesis, the [125I]gp71 binding level declined to approximately 20 to 30% of control values. During this period the rate of reduction of binding was very rapid and, in general was similar for both splenic and thymic cells. Further progression of the disease resulted in little or no further reduction in binding. The application of this technique to monitor host ecotropic virus synthesis and to study cell surface virus receptor control mechanisms in vivo is discussed.

Potentiation of leukemogenicity and infectivity of Rauscher leukemia virus by an enhancing factor present in egg fluids.

Sequential intraperitoneal administration of a low molecular-weight-enhancing factor, isolated from egg fluids, in mice infected with Rauscher leukemia virus (RLV) significantly stimulated viral replication. This was evidenced by elevated levels of viral DNA-polymerase in mouse sera. This stimulation of viral replication correlated with aggrevation of viral leukemogenicity, as reflected by increment of splenomegalic response and shortening of survival time. The in vivo potentiation of replication and leukemogenicity of RLV is achieved at least partly by the enhancing factor at the cellular level, since RLV replication was found to be stimulated also in an in vitro system. We assume that the stimulatory effect of the enhancing factor on viral replication is connected with its stimulatory effect on the synthesis of cellular DNA.

Mammalian retrovirus-associated RNase H is virus coded.

RNase H of a temperature-sensitive mutant of Rauscher murine leukemia virus is thermolabile, establishing this activity as a virus-coded function of the mammalian type C virus reverse transcriptase.

Enhanced production of Rauscher leukemia virus after infection of high-passaged JLS-V9 cells.

JLS-V9, a mouse bone marrow cell line infected with Rauscher leukemia virus at high passage level, produced larger amounts of virus than the standard JLS-V10 cells. The enhanced virus production was attributed to the increased saturation density of JLS-V9 cells.

Morphological conversion of 'immature' Rauscher leukaemia virus cores to a 'mature' form after addition of the P65-70 (gag gene product) proteolytic factor.

When the partially purified P65-70 proteolytic factor was added at increasing concentrations to 'immature' core sub-particles of Rauscher leukaemia virus (RLV), we observed an increased cleavage of P65-70 (the gag gene product) and P40 (an intermediate cleavage product containing p30) to p30, the major group specific antigen. When examined by electron microscopy the immature cores exhibited a linear decrease in number, with a concomitant increase in the number of mature cores after treatment. Various intermediate structures retaining elements of both immature and mature forms were also observed, suggesting that the in vitro conversion from immature cores to mature cores can occur on a I:I basis.

Relationship of retrovirus polyprotein cleavages to virion maturation studied with temperature-sensitive murine leukemia virus mutants.

Murine leukemia virus mutants ts3 (Moloney) and ts24 (Rauscher) both formed late-budding structures on the cell membrane at restrictive temperature. They both accumulated core polyproteins Pr65gag and Pr180gag-pol in cell membranes, but the envelope precursor was rapidly turned over. After shift to permissive temperature in the presence of cycloheximide, the accumulated precursors were sequentially cleaved via discrete intermediates both during the final stages of the budding process and in newly released virions to yield the finished virion core proteins and reverse transcriptase. The precursor form of reverse transcriptase was not enzymatically active and became activated partially or entirely inside released virions.