Synthesis of Rauscher murine leukemia virus-specific polypeptides in vitro.

The biosynthesis of specific polypeptides directed by purified viral messenger RNA from JLS-V9 cells infected with Rauscher leukemia virus has been studied in a rabbit reticulocyte lysate. The 35S viral mRNA gives rise to two major products of 65,000 and 72,000 molecular weight. The synthesis of specific polypeptides was also investigated in lysates derived from infected cells. The main products were polypeptides with molecular weights of 65,000, 76,000, and 82,000, and were preferentially made in association with membranes. The relative content of the virus-specific polypeptide of 65,000 molecular weight, synthesized in a cell-free system supplemented with purified polyribosomes, is considerably higher for membrane-bound polyribosomes.     

Effect of Rauscher leukemia development on DNA synthesis by hematopoietic CFU-S.

The effect of Rauscher viral leukemia on the number of colony forming cells (CFU-S) in the spleen has been measured as a function of time after virus administration. These were increased substantially after the first week, reaching a maximum at 2 weeks, after which they decreased in number. Determination of the proportion of these CFU-S which were in DNA synthesis was carried out using the in vitro 3HTdR suicide technique. Within 4 hours of injection of the virus the number of spleen CFU-S which could be inactivated by in vitro 3HTdR exposure increased to 20.9% from a normal mean value of 10.2%. For the next 2 weeks the proportion of CFU-S responding to tritiated thymidine exposure remained at the same value of approximately 20%; the change in number of surviving CFU-S roughly paralleled the change in total colony forming cells. In the third week after virus injection, the proportion of CFU-S which was inactivated by 3HTdR decreased to the point where it could no longer be detected, even though the total number of CFU-S was elevated several fold over the normal. These findings indicate that infection with moderate dose levels of Rauscher virus does result in a measurable early increase in the proportion of cells in DNA synthesis. However, the failure to detect further substantial increases as the disease develops suggests that the virus of itself is not an initiator of all the steps of DNA synthesis. Rather, it would appear that it is to some degree dependent upon other aspects of normal DNA synthesis which remain under control of the cell.     

Isolation of BPgp70, a fibroblast receptor for the envelope antigen of Rauscher murine leukemia virus.

A protein that avidly binds gp70, the envelope antigen of Rauscher murine leukemia virus (RMuLV), has been purified from the culture medium used for growth of BALB/c 3T3 mouse cells. Gel filtration chromatrography revealed the apparent Mr 10,000 BPgp70 was efficiently labeled when BALB/c 3T3 cells were grown in medium containing [3H]leucine, indicating a cellular origin for BPpg70. Metabolically labeled [3H]BPgp70 was not immunoprecipitated by IgG-anti RMuLV-gp&) alone, but was immunoprecipitated when gp70 was added, an indicaton of BPgp70 x gp70 complex formation. The dissociation constant estimated by immunoprecitipation agreed with the apparent Kd for binding of gp70 to BALB/c 3T3 cells. BPgp70 reversibly inhibited specific binding of 125I-labeled BMuLV-gp70 to BALB/c 3T3 cells when it was incubated with the 125I-labeled gp70 first. These data yielded a dissociation constant similar to that calculated from the immunoprecipitation data. 125I-Labeled BPgp70 also bound specifically to cells infected with RMuLV, but not to uninfected cells. Incubation of BALB/c 3T3 cells with the IgG fraction of an antiserum to BPgp70 inhibited the specific binding of 125I-labeled gp70 to these cells, but preimmune IgG did not. Complete inhibition was achieved at a less than 100:1 ratio of IgG anti-BPgp70 to gp70 binding sites.     

The influence of macrophages on the leukemogenic activity of Rauscher murine leukemia virus

A single application of trypan blue 3 to 24 hours before or simultaneous with inoculation of the Rauscher leukemia virus (RLV) resulted in enhancement of leukemogenesis with an especially high viremia. Repeated administration of trypan blue after RLV infection resulted in reduced spleen weight. Viremia, however, was also increased compared with controls (no application of trypan blue), but in a lower rate than in mice treated with the virus plus trypan blue. Since trypan blue is known as an inhibitor of the functions of macrophages the results point at an important role of this cell type in preventing infection with this oncogenic retrovirus. In this regard there are differences in the role of macrophages in human immunodeficiency virus (HIV) infections.     

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)     

Separation of sarcoma virus-specific and leukemia virus-specific genetic sequences of Moloney sarcoma virus.

We have studied the nucleic acid sequences in nonproducer cells transformed by Moloney sarcoma virus or Abelson leukemia virus (two types of replication-defective, RNA-containing, viruses isolated by passage of Moloney leukemia virus in BALB/c mice). DNA probes from the Moloney leukemia in virus detect RNA in both Abelson virus-transformed nonproducer cells and Moloney sarcoma virus-transformed nonproducer cells. A sarcoma-specific cDNA, prepared from the Moloney sarcoma virus, has extensive homology to RNA found in heterologous nonproducer cells transformed by Moloney sarcoma virus, has little homology to RNA in cells producing Moloney leukemia virus, and no detectable homology to RNA in nonproducer cells transformed by the Abelson virus. By analogy to earlier data on avian and mammalian sarcoma viruses, these results suggest that the Moloney sarcoma virus arose by recombination between a portion of the Moloney leukemia virus genome and additional sarcoma-specific information, and indicate that the expression of this information in not essential for Abelson virus-mediated fibroblast transformation.     

In vitro synthesis of repressible yeast acid phosphatase: identification of multiple mRNAs and products.

Antibodies to repressible nonspecific acid phosphatase [APase; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2] purified from Saccharomyces cerevisiae were used to detect the in vitro products of APase mRNA. Immunoprecipitation of cell-free synthesized protein and of in vivo enzyme from cell extracts has shown that derepression of enzyme synthesis in situ is the result of de novo appearance of functional mRNA followed by de novo protein synthesis. At least three unique APase polypeptides are synthesized in vitro from separate mRNAs and appear to be glycosylated in vivo to form secreted enzyme.     

The effect of Rauscher murine leukemia virus infection on the hemopoietic system of BALB/c mice. Cell proliferation and cell loss

Cell proliferation was investigated in normal and Rauscher Leukemia Virus-infected BALB/c mice. Five days after inoculation, islands of leukemic blasts arose in the red pulp, and proliferated as shown by autoradiographic analysis after a pulse of 3H-Thymidine. These cells subsequently infiltrated the whole spleen and 3 weeks after infection about 60% of the spleen consisted of large immature erythroblast-like cells. Repeated injections of 3H-Thymidine led to uniform labeling of 85% of the spleen cells. Cell cycle analysis showed that for bone marrow as well as for spleen cells the total duration of the cell cycle did not differ from the cell cycle times of normal erythroblasts. From the difference between the actual doubling time and the potential doubling time (estimated on the basis of the cell cycle time) it can be calculated that considerable cell loss must occur. This cell loss is only to a minor extent due to the release of blasts into the peripheral blood. Probably cell death and extrusion of nuclei during erythroid differentiation are the main factors involved.     

Identification of separate mRNAs coding for the alpha and beta subunits of thyrotropin.

A 9S mRNA, purified from mouse thyrotropic pituitary tumors by sucrose density gradient centrifugation of poly(A)-enriched mRNA, directed the synthesis of only alpha and beta subunits of thyrotropin in the reticulocyte lysate translation system. Analysis of radioiodinated 9S mRNA, repurified by oligo(dT)-cellulose chromatography and sucrose gradient centrifugation, yielded two species of RNA on urea/polyacrylamide gel electrophoresis. The major RNA species contained 620 nucleotides, and the minor RNA species contained 560 nucleotides. Unlabeled 9S mRNA was further purified by urea/polyacrylamide gel electrophoresis; the mRNAs were separately eluted from slices of the gel containing material migrating with an apparent length of 620 and 560 nucleotides. Translation of these mRNAs in the reticulocyte lysate showed that the longer mRNA coded for the alpha subunit and the shorter mRNA coded for the beta subunit of mouse thyrotropin. Because more alpha than beta subunit of thyrotropin was consistently synthesized, unbalanced amounts of thyrotropin subunits appear to be synthesized by translation of unbalanced amounts of individual mRNAs. We have demonstrated that the synthesis of thyrotropin is directed by two separate mRNA molecules, each coding for a different subunit of the hormone.     

Effect of a Rauscher leukemia virus vaccine upon chemical oncogenesis in the mouse.

The concept that type-C RNA viruses serve as determinants of chemically induced cancer would be supported if immunization against such viruses reduced the incidence of methylcholanthrene-induced sarcomas. A formalin vaccine was employed which was able to protect mice against the development of virus-induced Rauscher leukemia: 8/12 vaccinated mice survived versus 1/12 controls. When mice so immunized were challenged with near-threshold doses of chemical carcinogen, sarcoma incidence and death latency did not differ between vaccinated and control groups: within 10 months, a 320 mug dose of methylcholanthrene induced 100% sarcomas in both groups, while a 64 mug dose induced 62% and 65% tumors in vaccinated and control mice respectively. Thus, a relevant postulate of the oncogene hypothesis could not be supported by these studies. Formalin treatment neutralizes the oncogenic effect of mouse leukemia viruses yiwlding preparations that are immunogenic and can be successfully used as vaccines to protect against virus-induced leukemia (5). The finding of murine leukemia viruses in chemically induced tumors (1, 2) poses the question of whether these viruses are present in the tumors as passengers, or whether they are etiologically involved in the process of tumor induction. An approach to answering this question would be to challenge with chemical carcinogens mice which have been vaccinated with leukemia virus. If the virus were somehow involved in tumor induction, antiviral immunization might conceivably interfere with chemical carcinogenesis. Whitmire and Huebner (9) have reported that mice immunized with formalin-treated leukemia viruses, become resistant to sarcomagenesis by methylcholanthrene. Repetition of this experiment by Gericke and Chandra (6) gave non-significant differences between experimental and control groups. The present paper deals with the effect of immunization against Rauscher leukemia virus upon tumor induction by near-threshold doses of methylcholanthrene.     

Effect of antiserum on transplantable hematopoietic colony-forming units during Rauscher leukemia development.

Studies have been carried out to determine the sensitivity of hematopoietic CFU-S from Rauscher leukemic mice to an antiserum against the disease prepared in syngeneic mice. Test of this antiserum against Rauscher virus prior to injection showed it to be effective both in vitro and in vivo. At the same time, normal serum was shown to be without effect either against the CFU-S or against the virus. Spleen CFU-S were obtained from control and leukemic mice over a sequence of days following Rauscher virus injection and assayed by transplantation technique. Prior to transplantation these were incubated in vitro in either normal syngeneic serum or syngeneic antiserum. Incubation with antiserum had no effect on CFU-S obtained from the spleens of normal mice. However, incubation in this antiserum of spleen CFU-S from Rauscher leukemic mice resulted in a reduction of up to 50% in their colony-forming ability. Additional tests with guinea pig complement suggested that the levels of inactivation seen are not complement limited. This antiserum-induced reduction in colony formation was first evident in the second week after the injection of virus, coincident with the onset of splenomegaly in the leukemic mice. Thereafter, sensitivity of CFU-S to the antiserlm could be detected up to the terminal point of the leukemia (44 days).     

BLV-LB virus-specific sequences in cattle with spontaneous and experimentally induced leukemia.

Poly(A)-containing RNAs were extracted directly from the culture fluid of BLV-producing continuous and short-term lymphocyte cultures. The abovesaid poly(A)-RNAs were used as a template to synthesize complementary [3H]DNA (BLV cDNA). BLV cDNA possessed a high degree of homology to poly(A)-RNA isolated directly from the blood plasma of a leukemic animal. In contrast of leukemic cattle BLV-related sequences in white blood cells of normal animals were absent. Leukemic cells of cattle with both, spontaneous and experimentally induced leukemia contained bovine leukemia virus sequences in their genomes.     

Identification of the messenger RNAs coding for the gag and env gene products of the murine mammary tumor virus

Full-length (35S) genomic RNA from murine mammary tumor virus (MuMTV) was translated in vitro, using a reticulocyte lysate system, into proteins of 105,000, 75,000, 65,000, 35,000, and 27,000 daltons. These proteins were all immunoprecipitable with a monospecific antiserum to the major viral core protein, p27, but not with antiserum to the major viral envelope glycoprotein, gp47. Translation in vitro of RNA of about 24S size extracted from MuMTV yielded proteins similar in size and immunoreactivity to the products of the 35S RNA translation. Polyadenylylated RNA isolated from an MuMTV-producing cell line was fractionated according to size by velocity sedimentation and subsequently hybridized to MuMTV complementary DNA probes. These studies identified at least three size classes (35S, 24S, and 14-18S) of intracellular MuMTV-specific RNA. The 35S intracellular RNA was translated into MuMTV-specific proteins identical in size and immunoreactivity to the products of the virion-derived 35S RNA. On the other hand, translation of the intracellular 24S RNA fraction resulted in the synthesis of proteins, of which two (of about 70,000 daltons) could be immunoprecipitated with anti-gp47 serum, but not with anti-p27 serum. From these data we conclude that MuMTV core and envelope proteins are synthesized from two different mRNAs with approximate sizes of 35S and 24S, respectively. Our results also imply that the intracellular 24S mRNA is synthesized by a process more complex than simple cleavage of the 35S RNA.     

Identification of new gene products coded from X regions of human T-cell leukemia viruses.

Antibodies were raised against oligopeptides deduced from the nucleotide sequence in the conserved region located between env and the 3' long terminal repeat in human T-cell leukemia virus type I (HTLV-I) and type II (HTLV-II) to detect a protein coded from this region in virus-infected cells. Two of these antibodies precipitated a protein of 41 kilo-daltons in HTLV-I-infected cell lines and a protein of 38 kilo-daltons in HTLV-II-infected cells. The protein in HTLV-I-infected cells was precipitated by plasma from patients with adult T-cell leukemia but not by plasma from a normal adult. These results indicate that these proteins were translated from new coding regions (X) present in HTLV-I and HTLV-II.