Paul V. O'Donnell

Leukemogenic properties of AKR dualtropic (MCF) viruses: Amplification of murine leukemia virus-related antigens on thymocytes and acceleration of leukemia development in AKR mice☆

Abstract The late preleukemic period in AKR mice (6–8 months of age) is characterized by amplified expression of murine leukemia virus (MuLV)-related cell surface antigens on thymocytes. Analysis of thymic biopsies of preleukemic AKR mice at 180 days of age showed that antigen amplification was prognostic of spontaneous leukemia development within approximately 100 days. In the present study we have investigated the viral basis of this preleukemic change in AKR thymus. Cloned isolates of ecotropic, xenotropic, and dualtropic MuLV that were derived from preleukemic or leukemic AKR thymus were tested by intrathymic injection of young AKR mice to determine which of the three classes of MuLV could induce antigen amplification, and, subsequently, accelerate leukemia development in the same animal. Only dualtropic MuLV exhibited these two activities in vivo . Considerable heterogeneity was observed among the 13 dualtropic MuLV isolates examined. In vitro three distinct serological phenotypes could be recognized on the basis of expression of MuLV gag gene-coded antigens GCSA and MuLV env gene-coded antigens. G IX , G (ERLD) , G (RADAI) , G (ARSL2) on the surface of infected cells. Virus isolates also differed in (i) relative ecotropic-xenotropic host range (dualtropism) as assayed by ratios of infectious titers on mouse and mink cells, (ii) induction of mink cell foci (MCF), and (iii) infectivity for NIH/3T3 cells. In vivo the two activities of antigen amplification and leukemia acceleration could be dissociated and thus represent distinct virus phenotypes. Seven isolates of dualtropic MuLV induced antigen amplification; these viruses encoded G IX , G (ERLD) , and G (AKSL2) antigens, were MCF inducing, had SC-1/mink titer ratios ≥ 0.4, and were infectious for NIH/3T3 cells. Only three of these virus isolates (MCF 247, MCF 69L1, and MCF 13) accelerated leukemia development. Analysis of dose-response relationships and kinetics of virus-induced antigen amplification by MCF 69L1 virus implicated virus infection of thymocytes in the preleukemic change. Moreover, the level of MuLV antigen expression on thymocytes at approximately 1 month postinjection of MCF 69L1 virus correlated directly with development of early leukemia. It is apparent that leukemia development in young AKR mice which can be induced experimentally by intrathymic injection of cloned isolates of dualtropic MuLV exhibits the hallmarks of spontaneous disease in this strain and thus can serve as a useful model for study of thymic leukemogenesis in mice.

Structural domains of endogenous murine leukemia virus gp70s containing specific antigenic determinants defined by monoclonal antibodies

Abstract Eight distinct antigenic determinants, or epitopes (labeled a-h) were identified on endogenous ecotropic murine leukemia virus (MuLV) gp70s using a series of murine and rat monoclonal antibodies. These epitopes were characterized by their distribution patterns in a panel of cloned MuLVs, and by their localization in fragments of gp70 generated either by spontaneous breakdown of purified gp70, or by controlled proteolysis of gp70 in solubilized virions. A major 32K carboxy terminal fragment was formed which contained epitopes b, c, and f. This fragment also possessed the p15(E) disulfide linkage site, and contained approximately four complex (type 1) carbohydrate chains. An amino terminal 35K fragment contained epitopes a, d, g, and h, and possessed two glycosylated sites, including a site which occasionally retained an endoglycosidase H-sensitive oligosaccharide chain. A related 49K fragment was also obtained which included the entire 35K region and contained an additional sequence bearing epitope e. In a series of dual-tropic MCF-type viruses studied, only those epitopes located in the 32K fragment were ever retained, indicating that for these recombinant viruses at least a portion of that domain was derived from the ecotropic parent. A model is presented indicating the likely orientation of these fragments and their structural characteristics.

Selective neutralization of ecotropic murine leukemia virus by monoclonal antibodies: Localization of a site on the gp70 protein associated with ecotropism☆

Abstract A panel of nine monoclonal antibodies against the gp70 and p15(E) envelope proteins of MuLV were tested in parallel for their ability to bind to virus and neutralize infectivity. Although each of the antibodies bound to N-ecotropic MuLV, only antibody against the gp70 f epitope neutralized infectivity. These results were interpreted as evidence for a specific site on the gp70 protein of ecotropic MuLV, in proximity of the gp70 f epitope, which was responsible for the ecotropic-specific binding of virus to the surface of cells.

The GIX antigen of murine leukemia virus: an analysis with monoclonal antibodies.

Abstract The G IX antigen of murine retrovirus gp70 is a marker for virus replication in various cell types, as well as for T-cell differentiation and leukemogenesis in particular mouse strains. The serological definition of G IX depends on the cytotoxicity of a particular rat antiserum for thymocytes from strain 129 mice. We have found that two rat monoclonal antibodies, elicited by immunization with AKR ecotropic virus or AKR leukemia cells, co-type closely with G IX in several assay systems. Like G IX , the epitopes identified by the monoclonal antibodies are amplified on thymocytes during leukemogenesis in AKR mice. These antibodies are cytotoxic for G IX + leukemia cells and for fibroblasts infected with G IX + viral serotypes. Though absorbed by G IX + thymocytes of various strains, the antibodies are not cytotoxic for these cells. We ascribe the latter result to lower representation of antigenic sites on normal thymocytes, and postulate that more than one epitope may participate in classical G IX -mediated cytotoxicity. The monoclonal antibodies do not react with viral env products synthesized in the presence of the inhibitor of glycosylation, tunicamycin. Reactivity with the antibodies appears to require a stable configurational change in the env precursor protein coinciding with glycosylation, rather than direct participation of carbohydrate in the antigenic site. Thus subsequent enzymatic removal of carbohydrate chains in vitro does not alter reactivity with the antibodies.

Multiplicity-dependent kinetics of murine leukemia virus infection in Fv-1-sensitive and Fv-1-resistant cells

Abstract Viral growth curves of N- and B-tropic murine leukemia viruses (MuLVs) were measured at varying multiplicities of infection (m.o.i.) on Fv-1-sensitive and -resistant cells. Both Fv-1-dependent and Fv-1-independent effects on the appearance of progeny virus were observed. At low m.o.i., growth curves in productively infected Fv-1-resistant cells were identical to those in Fv-1-sensitive cells with a latent period of 24–30 hr; under these conditions the virus-releasing Fv-1-resistant cells are doubly infected and show growth kinetics similar to those of the singly infected Fv-1-sensitive cells. The length of the viral latent period in both Fv-1-sensitive and -resistant cells was inversely related to m.o.i. and appeared to correlate with the fraction of multiply infected cells in a given population. The average yield per infected cell increased stoichiometrically as a function of m.o.i.

Biological effects of a murine retrovirus carrying an activated N-ras gene of human origin

We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.

Amplified expression of murine leukemia virus(mulv)-coded antigens on thymocytes and leukemia cells of akr mice after infection by dualtropic (mcf) mulv.

Abstract Recently, we have shown that several isolates of recombinant, dualtropic (MCF) murine leukemia virus (MuLV) can induce amplification of MuLV gag and env gene-coded antigens on thymocytes of young AKR mice and, subsequently, can accelerate leukemia in these same animals. With respect to env gene-coded antigens, it is unclear whether antigen amplification represents expression of env gene products of the input virus on the surface of infected thymocytes or whether infection by MCF viruses induces the expression of other endogenous env gene sequences. Infection by antigenically marked viruses has allowed us to decide between these alternatives. We have found that AKR MCF isolates 69L1,13, and 247 can be distinguished serologically by specific patterns of reactivity with mouse and rat monoclonal antibodies that recognize 10 distinct epitopes (single antigenic determinants) of AKR ecotropic MuLV gp70 and p15(E) proteins. Accordingly, 1-monthold AKR mice were injected intrathymically with either MCF 69L1, MCF 13, or MCF 247 viruses and thymocytes of virus-injected mice were then assayed for amplification of specific epitopes at 32—36 days postinjection. Quantitative expression of MuLV antigens was determined by flow microfluorometry using a fluorescence-activated cell sorter. The patterns of reactivity with monoclonal antibodies which were originally determined for each virus in vitro were found to breed true on thymocytes infected in vivo . Moreover, the phenotype of virus recovered in vitro from virus accelerated leukemias was identical to that of the injected virus. Thus, the preleukemic change of MuLV antigen amplification appears to represent expression on thymocytes of env gene products encoded by the infecting MCF viral genome which continues to be expressed in the resulting leukemias without apparent changes in the recombinant env gene.

Endogenous murine leukemia virus-encoded proteins in radiation leukemias of BALB/c mice

Abstract To explore the role of endogenous retroviruses in radiation-induced leukemogenesis in the mouse, we have examined virus-encoded proteins in nine BALB/c leukemias by pulse-chase labeling procedures and serological typing with monospecific and monoclonal antibodies. The major gag precursor protein, Pr65 gag , was observed in all cases, but only three leukemias expressed detectable amounts of the glycosylated gag species, gP95 gag , or its precursor, Pr75 gag . No evidence was found for synthesis of gag -host fusion proteins. None of the leukemias released infectious xenotropic or dualtropic virus, but all nine expressed at least one env protein with xenotropic properties. In two instances a monoclonal antibody, 35 56 , which is specific for the MuLV G IX antigen, displayed a distinctive reactivity with this class of env protein, although this antibody is unreactive with replicating xenotropic viruses. An ecotropic/xenotropic recombinant env protein with the same 35 56 phenotype was observed in a leukemia induced by a strongly leukemogenic virus isolated from a BALB/c radiation leukemia.