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.     

Rmcf2, a xenotropic provirus in the Asian mouse species Mus castaneus, blocks infection by polytropic mouse gammaretroviruses

Cells from the Asian wild mouse species Mus castaneus are resistant to infection by the polytropic host range group of mouse gammaretroviruses. Two factors are responsible for this resistance: a defective XPR1 cell surface receptor for polytropic murine leukemia viruses (P-MLVs), and a resistance factor detectable only in interspecies hybrids between M. castaneus and mice with an XPR1 variant that permits infection by xenotropic MLVs (X-MLVs) as well as P-MLVs. This second novel virus resistance phenotype has been associated with expression of viral Env glycoprotein; Northern blotting with specific hybridization probes identified a spliced X-MLV env message unique to virus-resistant mice. These observations suggest that resistance is due to expression of one or more endogenous X-MLV envelope genes that interfere with infection by exogenous P-MLVs. M. castaneus contains multiple X-MLV proviruses, but serial backcrosses reduced this proviral content and permitted identification of a single proviral env sequence inherited with resistance. The resistance phenotype and the provirus were mapped to the same site on distal chromosome 18. The provirus was shown to be a full-length provirus highly homologous to previously described X-MLVs. Use of viral pseudotypes confirmed that this resistance gene, termed Rmcf2, prevents entry of P-MLVs. Rmcf2 resembles the virus resistance genes Fv4 and Rmcf in that it produces Env glycoprotein but fails to produce infectious virus; the proviruses associated with all three resistance genes have fatal defects. This type of provirus Env-mediated resistance represents an important defense mechanism in wild mouse populations exposed to endemic infections.     

Diverse wild mouse origins of xenotropic, mink cell focus-forming, and two types of ecotropic proviral genes

We analyzed wild mouse DNAs for the number and type of proviral genes related to the env sequences of various murine leukemia viruses (MuLVs). Only Mus species closely related to laboratory mice carried these retroviral sequences, and the different subclasses of viral env genes tended to be restricted to specific taxonomic groups. Only Mus musculus molossinus carried proviral genes which cross-reacted with the inbred mouse ecotropic MuLV env gene. The ecotropic viral env sequence associated with the Fv-4 resistance gene was found in the Asian mice M. musculus molossinus and Mus musculus castaneus and in California mice from Lake Casitas (LC). Both M. musculus castaneus and LC mice carried many additional Fv-4 env-related proviruses, two of which are common to both mouse populations, which suggests that these mice share a recent common ancestry. Xenotropic and mink cell focus-forming (MCF) virus env sequences were more widely dispersed in wild mice than the ecotropic viral env genes, which suggests that nonecotropic MuLVs were integrated into the Mus germ line at an earlier date. Xenotropic MuLVs represented the major component of MuLV env-reactive genes in Asian and eastern European mice classified as M. musculus molossinus, M. musculus castaneus, and Mus musculus musculus, whereas Mus musculus domesticus from western Europe, the Mediterranean, and North America contained almost exclusively MCF virus env copies. M. musculus musculus mice from central Europe trapped near the M. musculus domesticus/M. musculus musculus hybrid zone carried multiple copies of both types of env genes. LC mice also carried both xenotropic and MCF viral env genes, which is consistent with the above conclusion that they represent natural hybrids of M. musculus domesticus and M. musculus castaneus.     

Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: control by a single dominant locus on chromosome 1.

Although xenotropic murine leukemia viruses cannot productively infect cells of laboratory mice, cells from various wild-derived mice can support replication of these viruses. Although the virus-sensitive wild mice generally lack all or most of the xenotropic proviral genes characteristic of inbred strains, susceptibility to exogenous infection is unrelated to inheritance of these sequences. Instead, susceptibility is controlled by a single dominant gene, designated Sxv, which maps to chromosome 1. Sxv is closely linked to, but distinct from Bxv-1, the major locus for induction of xenotropic murine leukemia viruses in laboratory mice. Genetic experiments designed to characterize Sxv show that this gene also controls sensitivity to a wild mouse virus with the interference properties of mink cell focus-forming murine leukemia viruses, and that Sxv-mediated susceptibility to xenotropic murine leukemia viruses is restricted by the mink cell focus-forming virus resistance gene Rmcf. These data, together with genetic mapping of the mink cell focus-forming virus cell surface receptor locus to this same region of chromosome 1, suggest that Sxv may encode a wild mouse variant of the mink cell focus-forming virus receptor that allows penetration by xenotropic murine leukemia viruses.     

Characterization of a polytropic murine leukemia virus proviral sequence associated with the virus resistance gene Rmcf of DBA/2 mice

The DBA/2 mouse Rmcf gene is responsible for in vivo and in vitro resistance to infection by the polytropic mink cell focus-forming (MCF) virus subgroup of murine leukemia viruses (MLVs). Previous studies suggested that Rmcf resistance is mediated by expression of an interfering MCF MLV envelope (Env) gene. To characterize this env gene, we examined resistance in crosses between Rmcf(r) DBA/2 mice and Mus castaneus, a species that lacks endogenous MCF env sequences. In backcross progeny, inheritance of Rmcf resistance correlated with inheritance of a specific endogenous MCF virus env-containing 4.6-kb EcoRI fragment. This fragment was present in the DBA/2N substrain with Rmcf-mediated resistance but not in virus-susceptible DBA/2J substrain mice. This fragment contains a provirus with a 5' long terminal repeat and the 5' half of env; the gag and pol genes have been partially deleted. The Env sequence is identical to that of a highly immunogenic viral glycoprotein expressed in the DBA/2 cell line L5178Y and closely resembles the env genes of modified polytropic proviruses. The coding sequence for the full-length Rmcf Env surface subunit was amplified from DNAs from virus-resistant backcross mice and was cloned into an expression vector. NIH 3T3 and BALB 3T3 cells stably transfected with this construct showed significant resistance to infection by MCF MLV but not by amphotropic MLV. This study identifies an Rmcf-linked MCF provirus and indicates that, like the ecotropic virus resistance gene Fv4, Rmcf may mediate resistance through an interference mechanism.     

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.     

Receptor-binding domain of murine leukemia virus envelope glycoproteins.

The surface glycoprotein (SU) of murine leukemia viruses (MuLVs) comprises two domains connected by a proline-rich hinge. The interaction of MuLV particles with subgroup-specific cell surface receptors depends primarily on two variable regions (VRA and VRB) located in the amino-terminal domain. To delineate the minimal receptor-binding domains, we examined the capacity of soluble envelope fragments to compete with the entry of virus particles. Amphotropic, ecotropic, polytropic, and xenotropic truncated SUs were produced by inserting stop codons in the env gene of the 4070A, Friend, MCF247 and NZB MuLVs, respectively. These fragments, as well as full-length envelope glycoproteins, were stably expressed in cells bearing the corresponding receptor. Synthesis, posttranslational modifications, transport, and secretion of the env gene products were monitored by immunoprecipitation. Cells expressing the modified SUs or naive cells preincubated with SU-containing conditioned media were infected with different pseudotypes of a retroviral vector carrying a beta-galactosidase marker gene. Reduction of cell susceptibility to infection in the presence of SU was used as a measure of receptor occupancy. The results indicated that the amphotropic and ecotropic envelope amino-terminal domains contain all of the determinants required for receptor binding. In contrast, additional sequences in the proline-rich region were needed for efficient interaction of the polytropic and xenotropic amino-terminal domains with the receptors.     

Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.     

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.     

Characterization of a novel murine retrovirus mixture that facilitates hematopoiesis

A new virus previously arose in BALB/c females mated repeatedly to C57BL/6 (B6) males and then injected with fixed, activated B6 male spleen cells (V. S. Ter-Grigorov, O. Krifuks, E. Liubashevsky, A. Nyska, Z. Trainin, and V. Toder, Nat. Med. 3:37-41, 1997). In the present study, BALB/cJ mice inoculated with virus-containing plasma from affected mice developed splenomegaly, which was caused by increased numbers of Sca-1(+) Lin(-) hematopoietic stem cells (HSC) and their differentiated progeny. Biological and molecular analyses of a new virus revealed a mixture of murine leukemia viruses (MuLVs). These MuLVs comprised ecotropic and mink lung cell focus-forming (MCF) virus classes and are termed Rauscher-like MuLVs because they bear numerous similarities to the ecotropic and MCF viruses of the Rauscher MuLV complex but do not include a spleen focus-forming virus. The ecotropic virus component alone transferred some disease characteristics, while MCF virus alone did not. Thus, we have described a novel virus mixture, termed Rauscher-like MuLV, that causes an increase in hematopoiesis due to activation of pluripotent HSC. Experiments using mice and a protocol that replicated the pregnancy and immunization strategy of the original experiment demonstrated that endogenous BALB/c mouse ecotropic and xenotropic MuLVs are activated by these treatments. Emv1 was expressed in the spleens of multiparous mice but not in those of virgin mice, and Bxv1Emv1-pseudotyped MuLVs were recovered following injection of fixed, activated B6 cells. Thus, multiple pregnancies and allostimuli appear to have provided the signals required for activation of and recombination among endogenous viruses and could have resulted in generation of the Rauscher-like MuLV mixture.     

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.     

Cloning of endogenous murine leukemia virus-related sequences from chromosomal DNA of BALB/c and AKR/J mice: identification of an env progenitor of AKR-247 mink cell focus-forming proviral DNA

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.     

Amphotropic proviral envelope sequences are absent from the Mus germ line.

We derived an amphotropic murine leukemia virus (MuLV) type-specific probe for use in Southern blot hybridizations with cloned and genomic DNAs. A 133-base-pair RsaI-RsaI fragment from the 5' env region of the amphotropic viral isolate 4070A was subcloned into M13mp18 and radiolabeled in vitro. The probe detected the proviral DNAs in mink cells infected with seven different amphotropic MuLV isolates. The probe did not cross hybridize with the DNAs of molecular clones of ecotropic, mink cell focus-forming, or xenotropic MuLVs; nor did it anneal to the proviral DNAs of four xenotropic or six mink cell focus-forming viral isolates grown in mink cells. DNAs of 12 inbred laboratory mouse strains and more than 15 different wild mouse species and subspecies were examined for the presence of endogenous amphotropic env-related fragments. Amphotropic env-related sequences were found only in the DNAs of wild mice trapped in southern California in an area previously shown to harbor mice producing infectious amphotropic virus. Restriction enzyme analyses of DNAs from these mice showed that amphotropic sequences were not present as germ line copies but were the result of congenital or horizontal infection or both in this population. The DNAs of 11 various mammalian and avian species, including both natural predators of mice and squabs from the farms with virus-positive mice, lacked amphotropic envelope-related sequences.     

A multistep process of leukemogenesis in Moloney murine leukemia virus-infected mice that is modulated by retroviral pseudotyping and interference.

Mixed retroviral infections frequently exhibit pseudotyping, in which the genome of one virus is packaged in a virion containing SU proteins encoded by another virus. Infection of mice by Moloney murine leukemia virus (M-MuLV), which induces lymphocytic leukemia, results in a mixed viral infection composed of the inoculated ecotropic M-MuLV and polytropic MuLVs generated by recombination of M-MuLV with endogenous retroviral sequences. In this report, we describe pseudotyping which occurred among the polytropic and ecotropic MuLVs in M-MuLV-infected mice. Infectious center assays of polytropic MuLVs released from splenocytes or thymocytes of infected mice revealed that polytropic MuLVs were extensively pseudotyped within ecotropic virions. Late in the preleukemic stage, a dramatic change in the extent of pseudotyping occurred in thymuses. Starting at about 5 weeks, there was an abrupt increase in the number of thymocytes that released nonpseudotyped polytropic viruses. A parallel increase in thymocytes that released ecotropic M-MuLV packaged within polytropic virions was also observed. Analyses of the clonality of preleukemic thymuses and thymomas suggested that the change in pseudotyping characteristics was not the result of the emergence of tumor cells. Examination of mice infected with M-MuLV, Friend erythroleukemia virus, and a Friend erythroleukemia virus-M-MuLV chimeric virus suggested that the appearance of polytropic virions late in the preleukemic stage correlated with the induction of lymphocytic leukemia. We discuss different ways in which pseudotypic mixing may facilitate leukemogenesis, including a model in which the kinetics of thymic infection, modulated by pseudotyping and viral interference, facilitates a stepwise mechanism of leukemogenesis.     

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.     

Cell surface expression of the env gene polyprotein of dual-tropic mink cell focus-forming murine leukemia virus.

Differences have been observed in the kinetics of processing of the env gene polyprotein of ecotropic, xenotropic, and dual-tropic mink cell focus-forming (MCF) murine leukemia virus. In pulse-chase experiments, the env gene polyprotein of the dural-tropic MCF virus exhibits a marked increase in stability relative to that of either ecotropic or xenotropic virus. A comparison of cell surface expression of env gene products of ecotropic, xenotropic, and dual-tropic MCF murine leukemia virus has been made. Only gp70 is accessible to lactoperoxidase-catalyzed radioiodination of fibroblasts infected by ecotropic or xenotropic virus, whereas both gp70 and the env gene polyprotein are expressed on the surface of dual-tropic MCF virus-infected cells.     

Precise identification of endogenous proviruses of NFS/N mice participating in recombination with moloney ecotropic murine leukemia virus (MuLV) to generate polytropic MuLVs

Polytropic murine leukemia viruses (MuLVs) are generated by recombination of ecotropic MuLVs with env genes of a family of endogenous proviruses in mice, resulting in viruses with an expanded host range and greater virulence. Inbred mouse strains contain numerous endogenous proviruses that are potential donors of the env gene sequences of polytropic MuLVs; however, the precise identification of those proviruses that participate in recombination has been elusive. Three different structural groups of proviruses in NFS/N mice have been described and different ecotropic MuLVs preferentially recombine with different groups of proviruses. In contrast to other ecotropic MuLVs such as Friend MuLV or Akv that recombine predominantly with a single group of proviruses, Moloney MuLV (M-MuLV) recombines with at least two distinct groups. In this study, we determined that only three endogenous proviruses, two of one group and one of another group, are major participants in recombination with M-MuLV. Furthermore, the distinction between the polytropic MuLVs generated by M-MuLV and other ecotropic MuLVs is the result of recombination with a single endogenous provirus. This provirus exhibits a frameshift mutation in the 3' region of the surface glycoprotein-encoding sequences that is excluded in recombinants with M-MuLV. The sites of recombination between the env genes of M-MuLV and endogenous proviruses were confined to a short region exhibiting maximum homology between the ecotropic and polytropic env sequences and maximum stability of predicted RNA secondary structure. These observations suggest a possible mechanism for the specificity of recombination observed for different ecotropic MuLVs.     

Oncogenicity of AKR mink cell focus-inducing murine leukemia virus correlates with induction of chronic phosphatidylinositol signal transduction.

Naturally occurring recombinant murine leukemia viruses (MuLVs), termed mink cell focus-inducing (MCF) viruses, are the proximal leukemogens in spontaneous thymic lymphomas of AKR mice. The mechanism by which these viruses transform lymphocytes is not clear. Previous studies have implicated either integrational activation of proto-oncogenes, chronic autocrine immune stimulation, and/or autocrine stimulation of growth factor receptors (e.g., interleukin 2 receptors) via binding of the viral env glycoprotein (gp70) to these receptors. Any one of these events could also involve activation of second messenger signaling pathways in the cell. We examined whether infection with oncogenic AKR-247 MCF MuLV induced transmembrane signaling cascades in thymocytes of AKR mice. Cyclic AMP levels were not changed, but there was enhanced turnover of phosphatidylinositol phosphates, with concomitant increases in diacyglycerol and inositol 1,4,5-triphosphate. Thus, phospholipase C activity was increased. Protein kinase C activity was also elevated in comparison to that in uninfected thymocytes. The above events occurred in parallel with MCF expression in the thymus and were chronically maintained thereafter. No changes in phospholipid turnover occurred in an organ which did not replicate the MCF virus (spleen) or in thymocytes of AKR mice infected with a thymotropic, nononcogenic MCF virus (AKV-1-C36). Therefore, only the oncogenic MCF virus induced phosphatidylinositol signal transduction. Flow cytometric comparison of cell surface gp70 revealed that AKR-247 MCF virus-infected thymocytes expressed more MCF virus gp70 than did thymocytes from AKV-1-C36 MCF virus-infected mice, suggesting that certain threshold quantities of MCF virus env glycoproteins may be involved in this signaling. This type of signal transduction is not induced by stimulation of the interleukin 2 receptor but is involved in certain oncogene systems (e.g., ras and fms). Its chronic induction by oncogenic MCF MuLV may thus initiate thymocyte transformation.     

An Integrated Somatic Cell Hybrid, YAC, and BAC Map of the Rmc1 Region of Mouse Chromosome 1

Rmc1, the cellular receptor for the polytropic class of murine retroviruses, determines the tissue tropism of the virus and therefore plays a critical role in the pathogenesis of polytropic virus-induced leukemia. Previously we reported the physical mapping of this gene to a 5-cM region of mouse chromosome 1 and the construction of a yeast artificial chromosome (YAC) contig across this region. In this report we describe the refinement of the Rmc1 candidate region to approximately 600 kb and the generation of an integrated somatic cell hybrid, YAC, and bacterial artificial chromosome contig spanning the region. A number of genes and loci were physically ordered along the chromosome, including a recently identified candidate for Rmc1.