Rex Risser

Mechanism of interaction between endogenous ecotropic murine leukemia viruses in (BALB/c × C57BL/6) hybrid cells

The germline ecotropic murine leukemia (MuLV) proviruses of BALB/c and C57BL/6 (B6) mice were analyzed to determine the molecular basis of low virus expression in these mouse strains and to determine the mechanism of interaction of these two proviruses. Previous work had demonstrated that the BALB/c endogenous ecotropic provirus was infectious but unable to induce XC cell syncytia formation, and that induced (BALB/c X B6) hybrid cells expressed 10- to 50-fold more XC syncytia than induced parental cells. Two independently isolated DNA clones of the B6 endogenous ecotropic provirus were noninfectious following transfection into cells, and cell lines that expressed this viral genome produced noninfectious MuLV. Nucleotide sequencing of the mutant region of the B6 provirus indicated that the defective nature of this provirus resulted from an amino acid substitution of proline for alanine in the central portion of reverse transcriptase. From the analysis of the virus produced by induced hybrid cells, and the patterns of steady-state viral RNA in induced cells, we propose that the enhanced XC cell syncytia formation observed in hybrid cells is due to trans-complementation of viral proteins and not viral recombination or trans-activation of viral genome expression.

Genetic interactions in induction of endogenous murine leukemia virus from low leukemic mice

The frequency of ecotropic murine leukemia virus (MuLV) production in cells induced with halogenated pyrimidines has been investigated in several low leukemic strains of mice. Very few BALB/c or C57BL/6 (B6) induced embryo cells produce MuLV; this low frequency increases 10 to 50 fold in cells of the BALB/c x B6 F1 hybrid. Data from back-crosses of the F1 hybrid to each parent and from BALB/c x B6 recombinant inbred strains indicate that the phenotype of enhanced MuLV production results from interaction of two unlinked loci, dominant (+/+) alleles of which are carried by either parent. Genetic tests with BALB/c x B6 recombinant inbred strains confirm this two-locus model. The loci are designated Inc-1 and Inb-1 to signify their phenotypic detection by induction and the BALB/c or B6 strain of origin, respectively. Examination of hybrids of BALB/c and of B6 with other strains indicates that strains related in pedigree to BALB/c carry Inc-1, whereas those related to B6 carry Inb-1. Identification of genetic loci that specifically interact to enhance MuLV production after exposure to halogenated pyrimidines indicates the existence of mechanisms that regulate the induction or intracellular expression of endogenous MuLV.

Nucleotide conservation of endogenous ecotropic murine leukemia proviruses in inbred mice: Implications for viral origin and dispersal

Nucleotide sequence analysis of the ecotropic murine leukemia proviruses of AKR, BALB/c, and C57BL/6 mice indicated that these viral genomes differ from each other in less than 0.5% of their sequenced nucleotides, whereas they differ from the laboratory Moloney, Friend, or RadLV viruses or a partial ecotropic provirus found in wild mice by 8-22% of their sequenced nucleotides. The limited variation of endogenous ecotropic proviruses found in these common mouse strains indicates that few cycles of virus replication separated the introduction of the ecotropic endogenous retroviruses into the germlines of the progenitors of these now divergent mouse strains, and is consistent with the hypothesis that these common inbred strains were derived from a pool of very few mice, at least one of which was infected with an ecotropic murine leukemia virus. Ecotropic germline proviruses now found in common inbred mice most likely derive from germline reintegrations of the viral progeny of that initial single infection.

The Activity of an ABL-MYC Retrovirus in Fibroblast Cell Lines and in Lymphocytes

Evidence from many laboratories indicates that v-abl and c-myc oncogenes both can participate in the induction of B-cell neoplasia, and under appropriate conditions both may synergize in that process. The first indication of the interaction of these two oncogenes was the experiments of Potter et al (1973) in which it was found that infection of pristane-primed mice with Abelson murine leukemia virus (A-MuLV) greatly decreased the latent period for plasmacytoma development. Molecular analysis of those tumors indicated that most tumors expressed the v-abl oncogene product and contained a c-myc gene deregulated by chromosomal translocation (Ohno et al 1984) . Although c-myc deregulation has clearly been implicated in plasmacytoma development (Shen-Ong et al 1982; Adams et al 1983; Mushinski et al 1983), retroviruses that express v-myc or c-myc appear to induce tumors of the myeloid series of cells (Bambaugh et al 1985; Vennstrom et al 1984) . Moreover, infection with A-MuLV does not accelerate plasmacytoma development in Eμ-myc transgenic mice (Dyall-Smith et al 1988) . On the other hand, spleen cells from such mice can develop into plasmacytomas following infection with A-MuLV (Sugiyama et al 1989) . To investigate the interaction of v- abl and c-myc deregulated oncogenes under conditions where both are introduced simultaneously into cells we have constructed a virus that expresses both genes.

Factors affecting the frequency of transformation of rat embryo cells by simian virus 40

Abstract The transformation of primary rat cells into established cell lines by simian virus 40 has been monitored using the different restrictive assays of colony formation in sparse culture, dense colony or focus formation on a confluent cell sheet, and colony formation in semisolid medium. Primary embryonic rat cell cultures are considerably less susceptivle to infection and subsequent transformation than the established mouse 3T3 cell line or later in vitro passages of rat cells. These embryonic cells show a stage-specific susceptibility to transformation but not to infection with a maximum susceptibility achieved at the 15th to 16th days of gestation. All transformed cell lines derived by SV40 infection of primary rat cells express viral T antigen as detected by immunofluorescence, though they differ greatly in their plating efficiency in semisolid medium containing methycellulose. Only the assay of colony formation in semisolid medium selects directly for transformants which plate well in that medium while all assays appear to select for cell lines containing viral T antigen.

A Retrovirus Expressing v- abl and c- myc Induces Plasmacytomas in 100% of Adult Pristane-Primed BALB/c Mice

Intraperitoneal mineral oils such as pristane induce plasmacytomas in up to 60% of inbred BALB/c mice over the course of the period of two years ( Potter et al. 1984). The appearance of the tumors can be speeded up dramatically by intraperitoneal injection of Abelson murine leukemia virus (A-MuLV) (Potter et al. 1973). However, the incidence of plasmacytomas does not increase, in part because other tumors, pre-B cell lymphomas and myeloid tumors also arise in this setting. Molecular genetic studies of plasmacytomas have revealed that 100% of these tumors show deregulated expression of the c-myc proto-oncogene (Mushinski 1988). The universal deregulation of c-myc may be necessary, but it appears to be only one step in a multistep scenario leading to plasmacytomagenesis. Recombinant retroviral vectors offer the possibility of studying the cooperation of myc with other oncogenes. So far two oncogenes, in combination with myc, have accelerated plasmacytoma induction as compared to pristane alone. These oncogene combinations, myc+raf (Troppmair et al. 1989, Kurie et al. 1990) or myc+ras (Clynes et al. 1988), in form of retroviruses, accelerated plasmacytoma onset, but did not increase the tumor incidence. Since v-abl apparently cooperated well with deregulated c-myc in A-MuLV-induced tumors in pristane-primed mice, we predicted that a retrovirus which coexpressed v-abl and c-myc would also induce plasmacytomas expeditiously. Thus the protein-encoding portion of c-myc cDNA, under the control of the widely active thymidine kinase promotor, was inserted in the genome of A-MuLV (Largaespada et al. 1990) and injected intraperitonially, either helper-free or with Moloney Murine Leukemia Virus (MoMuLV) helper virus, into immunocompetent inbred BALB/c-AnPt mice which had previously recieved a single injection of 0.5 ml pristane. When ascites accumulated and contained tumor-like cells, the mice were sacrificed and their tumors excised for transplantation, histopathological studies and for DNA and RNA extraction.

The Pathogenesis of Tumors Induced by Helper Virus-Free Abelson Murine Leukemia Virus

The Abelson murine leukemia virus (A-MuLV) rapidly induces pre-B cell lymphomas in susceptible mice (Risser et al., 1978) and transforms bone marrow lymphocytes in vitro (Rosenberg and Baltimore, 1976) when inoculated as a complex of virions containing A-MuLV genomes and Moloney MuLY (M-MuLV) genomes. Recently, we have analyzed the activity of helper virus-free A-MuLV pools harvested from supernatants of the psi2 retroviral packaging cell line which had been transfected with a cloned A-MuLV P160 provirus (Green et al., 1987a). The availability of helper virus-free A-MuLV has allowed us to follow the progression of transformed cells in vivo without the complications that result from virus replication (Green et al., 1987b; Whitlock et al., 1983). Individual transformed cells recovered from A-MuLV(psi2) -infected mice were analyzed molecularly and biologically to determine which properties correlate with the progression of the transformed cell to a fully malignant cell.

Determinants of Abelson murine leukemia virus pathogenesis.

The Abelson murine leukemia virus (A-MuLV) is a rapidly transforming, defective retrovirus that induces pre-B cell tumors in susceptible mice (Abelson and Rabstein 1970, Scher and Siegler 1974, Rosenberg and Baltimore 1976, Risser et al 1978). It encodes the v-abl pl60 transforming protein, a tyrosine-kinase viral oncogene product. The v-abl oncogene was formed by the fusion of the Moloney MuLV (M-MuLV) gag gene with cellular c-abl sequences (Reynolds et al 1978, Witte et al 1978). The A-MuLV genome is a genetic recombinant of M- MuLV with the c-abl proto-oncogene (Wang et al 1984), and thus expression of the v-abl gene is controlled by the LTR derived from M-MuLV. Although M-MuLV induces predominantly T-cell tumors, A-MuLV induces pre-B cell tumors. The tissue tropism of M-MuLV is controlled by the LTR (Chatis et al 1983).

Monoclonal Antibody to Abelson Lymphoma Cells

The Abelson murine leukemia virus (A-MuLV) is a defective transforming retrovirus that rapidly induces a variety of hematopoietic neoplasms including those of B-celis when injected into mice (reviewed in Risser 1982; Rosenberg and Baltimore 1980). A-MuLV is a genetic recombinant between Moloney MuLV (M-MuLV) and a normal cellular proto-oncogene, c-abl (Goff et al., 1980). The strain of A-MuLV used here encodes a novel gag-abl fusion protein of 160 kd (Goff et al 1981; Grunwald et al 1982). This v-abl product is a phosphoprotein that will phosphorylate itself and other proteins on tyrosine residues in immuno-precipitates (Van de Ven et al 1980; Witte et al 1980; Sefton et al 1981). Using tumor regressor antiserum Witte et al (1979) identified a cellular protein of 150 kd (NCP150) in uninfected cells that is serologically related to the v-abl product; thus NCP150 is a candidate for the c-abl product. Ponticelli et al (1982) demonstrated that NCP150 shares 5 phosphopeptides with v-abl pl60. Wang and Baltimore (1983) demonstrated that c-abl mRNA was found in all normal cells and cell lines tested, though in highest quantities in mouse thymocytes and NIH 3T3 cells. We report here the isolation of a monoclonal antibody, 442, which reacts with normal protein(s) that have molecular properties reminiscent of those of the c-abl product or some receptors for growth factors (Cohen et al 1982).

THE ANOMALOUS ANTIBODY RESPONSE OF HYBRID MICE TO IMMUNIZATION WITH AN ABELSON VIRUS LYMPHOMA

ABSTRACT The humoral immune response of several hybrid mice to immunization with a parental Abelson virus lymphoma has been examined by serological and biochemical methods. Although all hybrid mice express the parental H-2 products of the immunizing tumor on their somatic cells, surprisingly they respond to the immunizing tumor with the production of cytotoxic and precipitating antibodies directed to those H-2 molecules. In all five positive responses, H-2K region products were recognized and in three cases H-2D region products were also recognized. Immunoprecipitation and gel electrophoresis of lactoperoxidase-radiolabe1 led cell surface molecules from normal spleen cells indicated the major reactivity was to molecules of approximate molecular weights 50,000 and 12,000 daltons, presumably the H-2K or D molecules plus beta-2-microglobulin. This “anti-self H-2 ” response of hybrid mice to immunization with this tumor may well reflect recognition of self in association with virus.