Naturally occurring murine leukemia viruses in wild mice: characterization of a new "amphotropic" class.

A new class of murine leukemia viruses, isolated from wild Mus musculus trapped in California, is described. These viruses, designated "amphotropic," replicate in mouse, rabbit, mink, human, guinea pig, and rat cells, but not in hamster, quail, or duck cells. They show N-tropism for mouse cells, and do not trigger the XC cell response. They are distinct by interference and virus neutralization testing from the previously recognized mouse-tropic and xenotropic MuLV classes. Mouse-tropic viruses occuring along with three of the four amphotropic isolates were found to be distinguishable by virus neutralization from other mouse-tropic murine leukemia virus strains of laboratory mouse origin.     

Characterization of recombinant nonecotropic murine leukemia viruses from the wild mouse species Mus spretus

The wild mouse species most closely related to the common laboratory strains contain proviral env genes of the xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs). To determine if the polytropic proviruses of Mus spretus contain functional genes, we inoculated neonates with Moloney MLV (MoMLV) or amphotropic MLV (A-MLV) and screened for viral recombinants with altered host ranges. Thymus and spleen cells from MoMLV-inoculated mice were plated on Mus dunni cells and mink cells, since these cells do not support the replication of MoMLV, and cells from A-MLV-inoculated mice were plated on ferret cells. All MoMLV-inoculated mice produced ecotropic viruses that resembled their MoMLV progenitor, although some isolates, unlike MoMLV, grew to high titers in M. dunni cells. All of the MoMLV-inoculated mice also produced nonecotropic virus that was infectious for mink cells. Sequencing of three MoMLV- and two A-MLV-derived nonecotropic recombinants confirmed that these viruses contained substantial substitutions that included the regions of env encoding the surface (SU) protein and the 5' end of the transmembrane (TM) protein. The 5' recombination breakpoint for one of the A-MLV recombinants was identified in RNase H. The M. spretus-derived env substitutions were nearly identical to the corresponding regions in prototypical laboratory mouse polytropic proviruses, but the wild mouse infectious viruses had a more restricted host range. The M. spretus proviruses contributing to these recombinants were also sequenced. The seven sequenced proviruses were 99% identical to one another and to the recombinants; only two of the seven had obvious fatal defects. We conclude that the M. spretus proviruses are likely to be recent germ line acquisitions and that they contain functional genes that can contribute to the production of replication-competent virus.     

Biochemical analysis of murine leukemia viruses isolated from radiation-induced leukemias of strain BALB/c.

Murine leukemia viruses isolated from radiation-induced BALB/c leukemias were characterized with respect to viral proteins and RNA. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the viral structural proteins revealed that for p12, p15, p30, and gp70, three of four electrophoretic variants of each could be detected. There was no correlation found between any of these mobilities and N- or B-tropism of the viruses. Proteins of all xenotropic viral isolates were identical in their gel electrophoretic profiles. The similar phenotypes of multiple viral clones from individual leukemias and of isolates grown in different cells suggest that the polymorphism of ecotropic viruses was generated in vivo rather than during in vitro virus growth. By two-dimensional fingerprinting of RNase T1-resistant oligonucleotides from 70S viral DNA, the previously reported association of N- and B-tropism with two distinct oligonucleotides was confirmed. The presence of two other oligonucleotides was correlated with positive and negative phenotypes of the virus-coded GIX cell surface antigen. The RNAs of two B-tropic isolates with distinctive p15 and p12 phenotypes differed from the RNA of a prototype N-tropic virus by the absence of three oligonucleotides mapping in the 5' portion (gag region) of the prototype RNA. In addition, one small-plaque B-tropic virus displayed extensive changes in the RNA sequences associated with the env region of the prototype.     

Biologic and molecular characterization of two newly isolated ras-containing murine leukemia viruses.

A murine sarcoma virus (MSV) was recovered from an (NFS X NS.C58v-1) F1 mouse which developed splenic sarcoma and erythroleukemia 6 months after inoculation with a mink cell focus-inducing murine leukemia virus (MuLV) isolated from an NFS mouse infected with a wild mouse ecotropic MuLV. The MSV, designated NS.C58 MSV-1, induced foci of transformation in mouse and rat fibroblasts, and inoculation of mice of various strains 2 weeks of age or younger resulted in erythroleukemia and sarcomatous lesions in spleen, lymph node, and brain. The MSV provirus was molecularly cloned from a genomic library prepared from transformed non-producer rat cells. The 8.8-kilobase proviral DNA contained a 1.0-kilobase p21 ras coding segment which replaced most of the gp70-encoding portion of an MuLV, most likely the endogenous C58v-1 ecotropic virus. The ras oncogene is closely related to v-Ha-ras by hybridization, expression of p21 protein, and nucleotide sequence. It is nearly identical in sequence to v-bas, the only previously described transduced, activated mouse c-ras. At position 12 in the p21 coding region, arginine is substituted for the naturally occurring glycine present in c-ras. A second MSV isolate is described which is similar to NS.C58 MSV-1 except for a 100- to 200-base-pair deletion in the noncoding region of the ras-containing insert.     

Acquisition of two endogenous ecotropic murine leukemia viruses in distinct Asian wild mouse populations.

Wild mouse DNAs were analyzed for two types of endogenous ecotropic murine leukemia viruses (MuLVs), Akv and Fv-4r-associated MuLV. Endogenous Akv viruses were found only in northern Chinese mice, Korean mice, and Japanese (Mus musculus molossinus) mice. The Fv-4r gene, which is a truncated endogenous MuLV with ecotropic interference properties, was carried by Southeast Asian (M. m. castaneus) mice, Korean mice, and M. m. molossinus. Sequences related to Fv-4r MuLV env were found only in M. m. castaneus. These findings suggest that endogenous Akv viruses were acquired by northern Chinese mice and that the Fv-4r gene or its related endogenous MuLVs were acquired independently by M. m. castaneus. The Fv-4r gene appears to have been generated hundreds of thousands of years ago, before the amplification of the Fv-4r-related endogenous MuLVs in M. m. castaneus. The coexistence of Akv viruses and the Fv-4r gene in M. m. molossinus may be explained by the hybrid origin of M. m. molossinus in crosses between northern Chinese mice and M. m. castaneus, as described in other articles. The absence of the Fv-4r-related endogenous MuLVs in M. m. molossinus may indicate that the ancestral mice of this subspecies either were an ancient type of M. m. castaneus that had acquired the Fv-4r gene but had not yet acquired the Fv-4r-related endogenous MuLVs or were a rare fraction of a mixed population of M. m. castaneus and northern Chinese mice.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.     

Lymphomas and high-level expression of murine leukemia viruses in CFW mice

Historically, Swiss Webster mice of the CFW subline, both inbred and random-bred stocks, have been considered to have a low spontaneous occurrence of hematopoietic system tumors, and previous reports of infectious expression of murine leukemia viruses (MuLVs) have been rare and unremarkable. In marked contrast, in the present study of CFW mice from one source observed by two laboratories over a 2-year period, nearly 60% developed tumors, 85% of which were lymphomas, the majority of B-cell origin. All tumors tested expressed ecotropic MuLVs, and most expressed mink cell focus-inducing (MCF) MuLVs. Among normal mice of weanling to advanced age, over one-half were positive for ecotropic virus in tail or lymphoid tissues, and MCF virus was frequently present in lymphoid tissue, less often in tail. Patterns of ecotropic proviral integration indicated that natural infection occurred by both genetic and exogenous routes. Lymphomas were induced in NIH Swiss mice infected as neonates with tissue culture-propagated MuLVs isolated from normal and tumor tissue of CFW mice.     

Expression of murine leukemia viruses in B-cell lymphomas of CWD/Agl mice.

The inbred mouse strain CWD/Agl has a high incidence of spontaneous B-cell lymphomas characterized by gross splenomegaly and lymph node enlargement. The endogenous ecotropic retrovirus of CWD/Agl mice is expressed in the spleen within the first 2 weeks of age and in the thymus by 1 month of age. Endogenous xenotropic virus is expressed in the spleen and bone marrow of the earliest age group examined (4 months). Restriction enzyme analysis of DNA extracted from tumorous tissues suggests that mink cell focus-forming viruses are not required for B-cell lymphomagenesis in CWD/Agl mice. CWD/Agl mice provide an important new experimental model for the study of B-cell lymphoma.     

Macrophage accumulation in mice is inhibited by low molecular weight products from murine leukemia viruses

Low m.w. extracts from three known oncogenic viruses, Friend, Moloney, and Rauscher, inhibited the accumulation of macrophages at sites of delayed inflammatory reactions in mice. The potential biologic significance of these proteins is suggested by their potency: as little as 1.2 ng of viral protein inhibited (p less than 0.02) macrophage accumulation when injected at a site distant to the inflammatory reaction. A virus envelope protein fraction of 15,000 daltons (p15E) was likewise found to inhibit macrophage accumulation and may in part represent the active factor of the virus extracts. Certain oncogenic viruses may thus exert their immunosuppressive activity by release of potent inhibitors of systemic macrophage function.     

Different genes control the susceptibility of mice to Moloney or Abelson murine leukemia viruses.

The susceptibility of mice to lymphoma induction by Moloney or Abelson murine leukemia virus has been compared in BALB/c, C57BL/6, and BALB/cXC57BL/6 recombinant inbred strains. BALB/c mice were found to be susceptible to lymphoma induction by either virus, and C57BL/6 mice were found to be relatively resistant to lymphoma induction by either virus. The genes that control these patterns of susceptibility to each virus are not the same because susceptibility to each virus segregated independently in CXB recombinant inbred strains. We also found, as reported by Cook (W. Cook, Proc. Natl. Acad. Sci. U.S.A. 79:2917-2921, 1982), when injected intrathymically that Abelson murine leukemia virus rapidly induced thymomas in weanling B6 mice. Examination of the cellular phenotypes of the tumors induced by Abelson murine leukemia virus or by Moloney murine leukemia virus indicated that different lymphocyte subpopulations were the targets for tumor induction by each virus.     

Genetic basis for resistance to polytropic murine leukemia viruses in the wild mouse species Mus castaneus.

Cultured cells derived from the wild mouse species Mus castaneus were found to be uniquely resistant to exogenous infection by polytropic mink cell focus-forming (MCF) murine leukemia viruses (MuLVs). This MCF MuLV resistance is inherited as a genetically recessive trait in the progeny of F1 crosses between M. castaneus and MCF MuLV-susceptible laboratory mice. Examination of the progeny of backcrosses demonstrated that susceptibility is inherited as a single gene which maps to chromosome 1. The map location of this gene places it at or near the locus Rmc1, the gene encoding the receptor for MCF/xenotropic MuLVs, suggesting that resistance is mediated by the M. castaneus allele of this receptor.     

Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.     

Isolation of paralysis-inducing murine leukemia viruses from Friend virus passaged in rats.

Four clones of murine leukemia viruses (PVC-111, PVC-211, PVC-321, and PVC-441) were isolated from a paralyzed Fischer rat which had been infected with rat-passaged Friend leukemia virus. PVC-211 and PVC-321 viruses induced hind leg paralysis in rats and killed them within 1 month, and PVC-441 did so within 2 months after infection, whereas PVC-111 did not within 4 months. PVC-321 and PVC-441 but not PVC-111 virus grew well in brain and spinal cord media. The viral antigens were found often in glia cells and rarely in neurons of the rats infected with each of these PVC viruses. All of the PVC viruses induced neuronal degeneration but neither inflammation nor leukemic infiltration in the spinal cord. The isolated viruses were all ecotropic and NB-tropic. Age dependency of the susceptibility of rats to paralysis induction was observed.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.