Dualtropic Murine Leukemia Virus Research Articles

Properties of the naturally occurring soluble surface glycoprotein of ecotropic murine leukemia virus: binding specificity and possible conformational change after binding to receptor.

Ecotropic murine leukemia virus (MuLV) infection is initiated by the interaction between the surface glycoprotein (SU) of the virus and its cell-surface receptor mCAT-1. We investigated the SU-receptor interaction by using a naturally occurring soluble SU which was encoded by the envelope (env) gene of a defective endogenous MuLV, Fv-4(r). Binding of the SU to mCAT-1-positive mouse cells was completed by 1 min at 37 degrees C. The SU could not bind to mouse cells that were persistently infected by ecotropic MuLVs (but not amphotropic or dualtropic MuLVs) or transfected with wild-type ecotropic env genes or a mutant env gene which can express only precursor Env protein that is restricted to retention in the endoplasmic reticulum. These cells were also resistant to superinfection by ecotropic MuLVs. Thus, superinfection resistance correlated with the lack of SU-binding capacity. After binding to the cells, the SU appeared to undergo some conformational changes within 1 min in a temperature-dependent manner. This was suggested by the different properties of two monoclonal antibodies (MAbs) reactive with the same C-terminal half of the Fv-4(r) SU domain, including a proline-rich motif which was shown to be important for conformation of the SU and interaction between the SU and the transmembrane protein. One MAb reacting with the soluble SU bound to cells was dissociated by a temperature shift from 4 to 37 degrees C. Such dissociation was not observed in cells synthesizing the SU or when another MAb was used, indicating that the dissociation was not due to a temperature-dependent release of the MAb but to possible conformational changes in the SU.

A Friend virus mutant that overcomes Fv-2rr host resistance encodes a small glycoprotein that dimerizes, is processed to cell surfaces, and specifically activates erythropoietin receptors.

The env gene of Friend spleen focus-forming virus (SFFV) encodes a membrane glycoprotein (gp55) that is inefficiently (3 to 5%) processed from the rough endoplasmic reticulum to form a larger dimeric plasma membrane derivative (gp55p). Moreover, the SFFV env glycoprotein associates with erythropoietin receptors (EpoR) to cause proliferation of infected erythroblasts [J.-P. Li, A. D. D'Andrea, H. F. Lodish, and D. Baltimore, Nature (London) 343:762-764, 1990]. Interestingly, the mitogenic effect of SFFV is blocked in mice homozygous for the Fv-2r resistance gene, but mutant SFFVs can overcome this resistance. Recent evidence suggested that these mutants contain partial env deletions that truncate the membrane-proximal extracellular domain of the encoded glycoproteins (M. H. Majumdar, C.-L. Cho, M. T. Fox, K. L. Eckner, S. Kozak, D. Kabat, and R. W. Geib, J. Virol. 66:3652-3660, 1992). Mutant BB6, which encodes a gp42 glycoprotein that has a large deletion in this domain, causes erythroblastosis in DBA/2 (Fv-2s) as well as in congenic D2.R (Fv-2r) mice. Analogous to gp55, gp42 is processed inefficiently as a disulfide-bonded dimer to form cell surface gp42p. Retroviral vectors with SFFV and BB6 env genes have no effect on interleukin 3-dependent BaF3 hematopoietic cells, but they cause growth factor independency of BaF3/EpoR cells, a derivative that contains recombinant EpoR. After binding 125I-Epo to surface EpoR on these factor-independent cells and adding the covalent cross-linking reagent disuccinimidyl suberate, complexes that had immunological properties and sizes demonstrating that they consisted of 125I-Epo-gp55p and 125I-Epo-gp42p were isolated from cell lysates. Contrary to a previous report, SFFV or BB6 env glycoproteins did not promiscuously activate other members of the EpoR superfamily. Although the related env glycoproteins encoded by dualtropic murine leukemia viruses formed detectable complexes with EpoR, strong mitogenic signalling did not ensue. Our results indicate that the SFFV and BB6 env glycoproteins specifically activate EpoR; they help to define the glycoprotein properties important for its functions; and they strongly suggest that the Fv-2 leukemia control gene encodes an EpoR-associated regulatory factor.

Fv-4 resistance gene: a truncated endogenous murine leukemia virus with ecotropic interference properties

Fv-4 is a mouse gene which controls susceptibility to infection by ecotropic murine leukemia virus (MuLV). We previously cloned part of an endogenous MuLV associated with the resistance allele of the Fv-4 gene (Fv-4r). In this report, we describe an extended clone of the Fv-4r allele consisting of a 17-kilobase DNA fragment containing the retroviral sequence and its 5'-flanking sequence. The new DNA clone contains a truncated MuLV with delta pol-env-long terminal repeat sequences but no other MuLV-reactive sequence within 13 kilobases upstream of the truncated MuLV. Transfection of this clone into mouse cells led to transcription of Fv-4 env mRNA, expression of the Fv-4r-specific MuLV envelope protein, and resistance to infection with ecotropic MuLV but not amphotropic and dualtropic MuLVs. Restriction of ecotropic viruses appears to occur at or before viral cDNA synthesis. This result is consistent with a model of receptor interference for Fv-4 restriction. Our data also suggest that the 5' non-MuLV sequence is important for biological function, since a DNA clone which lacks most of the 5'-flanking sequence did not efficiently confer the resistance phenotype.

The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity

A cell membrane “gp70” associated with Fv-4 gene: Immunological characterization, and tissue and strain distribution

Fv-4 r is a dominant resistance gene which controls susceptibility of mice to exogenous infection with ecotropic murine leukemia virus (MuLV). A MuLV env-gene product related to gp70 on cells from uninfected Fv-4 r mice has been found. Expression of this molecule is closely linked to the resistance allele. Antigenic determinants on the Fv-4 r gp70 are similar to many ecotropic and some dualtropic MuLV gp70s but different from xenotropic and amphotropic MuLV gp70s. The Fv-4 r gp70 molecule also has unique antigenic determinants since it is recognized by Fv-4 specific antisera which do not recognize other cell membrane gp70s or viral gp70s. The Fv-4-specific cell membrane gp70 is detectable on cells from thymus, spleen, lymph node, bone marrow, and embryo fibroblasts from Fv-4 r mice, but not from Fv-4 s mice. Slight differences in electrophoretic mobility of the Fv-4 r gp70 are found on different tissues. This unique cell membrane gp70 may be causally related to Fv-4 resistance.

Reduced leukemogenicity caused by mutations in the membrane glycoprotein gene of Rauscher spleen focus-forming virus.

We isolated and characterized two spontaneous, weakly leukemogenic mutants of Rauscher spleen focus-forming virus (R-SFFV) that contain mutations in nonoverlapping regions of the membrane envelope (env) glycoprotein gene. As reported previously (M. Ruta and D. Kabat, J. Virol. 35:844-853, 1980), the replication-defective R-SFFV encodes a membrane glycoprotein with an apparent Mr of 54,000 (gp54) which is structurally and immunologically related to the membrane envelope glycoproteins of dual-tropic murine leukemia viruses. Mutant R-SFFV clones 3-25 and 4-3 encode abnormally sized gp54-related glycoproteins with apparent Mrs of 52,000 (gp52) and 45,000 (gp45), respectively. Northern and Southern blot analyses of the mutant R-SFFV nucleic acids indicated that an insertion has occurred in the 3-25 env gene and that a deletion has occurred in the 4-3 env gene. Furthermore, restriction endonuclease analyses and comparisons of the fragmentation patterns of the wild-type and mutant glycoproteins generated by partial proteolysis with Staphylococcus aureus V8 protease indicated that the mutations affect nonoverlapping domains of the envelope glycoprotein (amino-terminal fragment affected in 3-25 glycoprotein and carboxyl-terminal fragment affected in 4-3 glycoprotein). Glycosylation inhibition studies indicated that the reduced size of gp52 is caused at least partly by loss of an asparagine-linked oligosaccharide. In addition, these mutant viruses have dramatically reduced leukemogenicities compared with wild-type R-SFFV. We conclude that the gp54 structural gene is required for initiation or amplification of the splenic erythroblast hyperplasia which characterizes the preleukemic phase of Rauscher disease.

Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s.

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Genetic structure of Rauscher spleen focus-forming virus.

Rauscher spleen focus-forming virus contains functional gag and pol genes and a partially deleted env gene which is structurally related to the env genes of dual tropic murine leukemia viruses.

Amplified expression of murine leukemia virus (MuLV)-coded antigens on thymocytes and leukemia cells of AKR mice after infection by dualtropic (MCF) MuLV

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.

Leukemogenesis by Gross passage A murine leukemia virus: expression of viruses with recombinant env genes in transformed cells.

Gross passage A murine leukemia virus (MuLV) derived from extracts of C3Hf/Bi mouse leukemias has been shown to be a virus complex consisting of ecotropic, xenotropic, and recombinant, dualtropic MuLV components. The three virus components were distinguished biochemically by differences in the molecular weights and peptide maps of their primary env gene products synthesized in infected cells in vivo and in vitro. Virus expression was studied in primary leukemias induced in C3Hf/Bi mice by Gross passage A virus extracts and by the individual ecotropic and recombinant MuLV components that were isolated in vitro. Our findings suggest that expression of the recombinant MuLV component of the Gross passage A virus complex is necessary and sufficient for the induction of leukemias in C3Hf/Bi mice. In contrast, induction of leukemias by the ecotropic virus component appears to involve generation of a second virus with characteristics of recombinant, dualtropic MuLV.

A cell surface antigen of the mouse related to xenotropic MuLv defined by naturally occurring antibody and monoclonal antibody. Relation to Gix G(rada1), G(aksl2) systems of MuLV-related antigens.

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.

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

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.

Serological analysis of antigenic determinants on the env gene oroducts of AKR dualtropic (MCF) murine leukemia viruses

A panel of eight monoclonal antibodies recognizing individual antigenic determinan (epitopes) on the envelope proteins (gp70 and p15(E)) of murine leukemia virus (MuLV) has been used to probe the serological properties of recombinant dualtropic (MCF) viruses. Since six of these monoclonal antibodies showed specificity for epitopes that were exclusive to endogenous N-ecotropic MuLV, it was reasoned that they might provide a means to determine the relative genetic contribution of ecotropic information in the env gene of dualtropic isolates. It was anticipated that the loss of ecotropic-specific sequences in these viruses would be reflected by a loss of reactivity with the ecotropic-specific monoclonal antibodies. This expectation proved to be correct, as several of the monoclonal antibodies that reacted with endogenous ecotropic MuLV of AKR mice failed to react with isolates of recombinant dualtropic MuLV from the same strain. In addition, it was noted that the “nonreactivity” of the monoclonal antibodies with different dualtropic viruses occurred in characteristic patterns. A common feature of the patterns was coordinate loss of expression of the gp70 a, gp70 d, and gp70 e epitopes from all AKR dualtropic MuLV. In contrast to the common deletion of ecotropic-specific epitopes, the gp70 b, gp70 c, and p15(E) a epitopes were variably deleted in different recombinant viruses.

Genomic masking and rescue of dual-tropic murine leukemia viruses: role of pseudotype virions in viral lymphomagenesis.

The kinetics of genomic masking of nondefective dual-tropic murine leukemia viruses (MuLV) by ecotropic MuLV in mixedly infected mouse cells was studied. The ratio of virus infection (ecotropic to dual-tropic) determined the kinetics of genomic masking. Some dual-tropic virus isolates could be masked routinely (i.e., converted to virions containing a dual-tropic genome and possessing the ecotropic host range) at all ratios of initial infection of mixedly infected mouse cells. The masked genomes could be rescued as infectious viruses of dual-tropic genotype and host range by an infectious center assay of the infected mouse cells on mink lung cells. Infectious center rescue of masked dual-tropic MuLV took place readily, even from cells that had been kept in continuous culture for many months after the onset of genomic masking. Some dual-tropic virus clones did not undergo genomic masking at any infection ratio with ecotropic virus. Nevertheless, such mixedly infected cultures also gave rise to phenotypically mixed virions, which contained a dual-tropic genome and had an ecotropic host range. (The phenotypically mixed virions found among the progeny of mixedly infected mouse cells were not pseudotypes, as both types of viruses were genetically nondefective, nor was the process leading to their generation a bona fide phenotypic mixing [Fischinger et al., Science 201:457-459, 1978]. Nevertheless, in this paper we use the terms pseudotype and phenotypic mixing because of the lack of a better description.) The lymphomagenic potential of dual-tropic lymphomagenic MuLV was compared with that of phenotypically mixed virions possessing an ecotropic host range and with that of a simple mixture of dual-tropic and ecotropic viruses. The phenotypically mixed pseudotype virions were more potent lymphoma inducers than were those of dual-tropic, cloned genotype. Inoculation of a simple mixture of the viruses did not increase dual-tropic virus tumorigenicity. The reason for this was probably the highly efficient inactivation of dual-tropic virus by oncovirus-inactivating factor, which is present in normal mouse serum and did not inactivate the phenotypically mixed virions. Simple mixtures of dual-tropic lymphomagenic and ecotropic virus preparations behaved like the cloned, dual-tropic virus in vivo and were equally sensitive to oncovirus-inactivating factor in vitro. Thus, phenotypic mixing of dual-tropic and ecotropic MuLV with or without concomitant genomic masking may be a highly significant phenomenon in naturally occurring lymphomagenesis. It may also be important to use phenotypically mixed viruses in the procedures used for in vivo testing of lymphomagenic dual-tropic MuLV isolates.

Inhibition of AKR leukemogenesis by SMX-1, a dualtropic murine leukemia virus.

Intrathymic injection of SMX-1, a dualtropic murine leukemia virus (MuLV) originally derived from Moloney murine leukemia virus stocks, protects AKR mice from developing MuLV-accelerated leukemia and spontaneous leukemia. Thymuses of SMX-1-injected mice show no change in weight, morphology, or thymocyte size, and quantitative expression of Thy-1 and Lyt-2 differentiation antigens is identical to control mice. The amplified thymic expression of MuLV-related antigens that occurs spontaneously in 6-month-old preleukemic AKR mice or that can be induced in young AKR mice by leukemogenic AKR dualtropic MuLV is prevented by SMX-1. It appears unlikely that the protective effect of SMX-1 is explicable in terms of cross-immunogenicity with transforming MuLV or transformed cells. As SMX-1 persists for long periods after intrathymic injection and does not alter levels of thymic ecotropic MuLV, SMX-1 may interfere with the generation, spread, or leukemogenicity of dualtropic MuLV that form de novo in AKR thymus during the late preleukemic phase. SMX-1 provides a way to probe the events leading to cell transformation in AKR mice.