Generation of novel syncytium-inducing and host range variants of ecotropic moloney murine leukemia virus in Mus spicilegus

Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SUenv. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni.     

Structures of endogenous nonecotropic murine leukemia virus (MLV) long terminal repeats in wild mice: implication for evolution of MLVs

To develop a better understanding of the interaction between retroviruses and their hosts, we have investigated the polymorphism in endogenous murine leukemia proviruses (MLVs). We used genomic libraries of wild mouse DNAs and PCR to analyze genetic variation in the proviruses found in wild mouse species, including Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus), Mus spretus, and Mus spicelegus, as well as some inbred laboratory strains. In this analysis, we detected several unique forms of sequence organization in the U3 regions of the long terminal repeats of these proviruses. The distribution of the proviruses with unique U3 structures demonstrated that xenotropic MLV-related proviruses were present only in M. musculus subspecies, while polytropic MLV-related proviruses were found in both M. musculus and M. spretus. Furthermore, one unique provirus from M. spicelegus was found to be equidistant from ecotropic provirus and nonecotropic provirus by phylogenetic analysis. This provirus, termed HEMV, was thus likely to be related to the common ancestor of these MLVs. Moreover, an ancestral type of polytropic MLV-related provirus was detected in M. spretus species. Despite their "ancestral" phylogenetic position, proviruses of these types are not widespread in mice, implying more-recent spread by infection rather than inheritance. These results imply that recent evolution of these proviruses involved alternating periods of replication as virus and residence in the germ line.     

Mink epithelial cell killing by pathogenic murine leukemia viruses involves endoplasmic reticulum stress

We previously demonstrated that mink cells undergo apoptosis after MCF13 murine leukemia virus (MLV) infection. In this study, we observed that virus-infected mink epithelial cells had significantly larger amounts of steady-state levels of MCF13 MLV envelope precursor protein (gPr80(env)) than did Mus dunni fibroblasts, which are resistant to virus-induced cytopathicity. Infection of mink cells with the noncytopathic NZB-9 MLV did not result in the accumulation of gPr80(env). MCF13 MLV infection of mink cells produced low cell surface expression of envelope glycoprotein and less efficient spread of infectious virus. Western blot analysis of mink epithelial cells infected with MCF13 MLV showed an increase in GRP78/BiP, which was not observed for either mink cells infected with NZB-9 MLV or M. dunni fibroblasts infected with MCF13 MLV. MCF13 MLV infection of mink cells also resulted in a significant upregulation of CHOP/GADD153. These results indicate that the accumulation of MCF13 MLV gPr80(env) triggers endoplasmic reticulum stress, which may mediate apoptosis in mink epithelial cells.     

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.     

A Mus dunni cell line that lacks sequences closely related to endogenous murine leukemia viruses and can be infected by ectropic, amphotropic, xenotropic, and mink cell focus-forming viruses

A Mus dunni cell line has been developed that is permissive for all four classes of murine leukemia viruses (MuLV): ecotropic, amphotropic, xenotropic, and mink cell focus-forming viruses. The M. dunni cells contain fewer MuLV-related sequences than do feral or domestic mouse, rat, or mink cells. Infection of the line by ecotropic MuLV induces a distinct cytopathic effect, and the cells can be readily transfected by MuLV DNA. The M. dunni line has been used to isolate an endogenous MuLV from the SC-1 feral mouse cell line.     

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.     

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.     

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.     

Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.     

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.     

Murine retroviruses use at least six different receptors for entry into Mus dunni cells.

Murine retroviruses have been divided into six interference groups that use different receptors for cell entry: the ecotropic, xenotropic, polytropic, amphotropic, 10A1, and Mus dunni endogenous virus groups. Some interference is observed between xenotropic and polytropic viruses and between amphotropic and 10A1 viruses, indicating some overlap in receptor specificity between these groups, but otherwise these interference groups appear completely independent. In contrast, one study found interference among many of these groups when Mus dunni wild mouse cells were examined with an immunofluorescence assay to detect infection by the challenge virus. Here we have used a more direct assay for cell entry by using pseudotyped retroviral vectors to measure interference in M. dunni cells, and we find no evidence for extensive interference between members of different murine retrovirus groups. Indeed, our results in M. dunni cells are consistent with interference results observed in other cell types and indicate that the anomalous interference results previously observed in M. dunni cells with the immunofluorescence assay were most likely due to factors other than those that affect receptor-mediated virus entry. In summary, our results show that murine retroviruses use at least six different receptors for entry into M. dunni cells.     

Somatically acquired recombinant murine leukemia proviruses in thymic leukemias of AKR/J mice.

We have probed the structure and arrangement of murine leukemia virus genomes in eight spontaneous AKR thymic leukemias by Southern hybridization with one ecotropic pol and four ecotropic env probes. These probes revealed many (in 2 cases over 15) somatically acquired proviruses that had undergone complex patterns of recombination. The large majority were not deleted and were structurally analogous to the oncogenic mink cell focus-inducing murine leukemia viruses isolated from AKR tumors in that the amino-terminal p15E-coding region derived from ecotropic AKR murine leukemia virus sequences, whereas certain gp70-coding sequences were nonecotropic. Nevertheless, we observed a few proviruses which did not appear to be gp70 recombinants; however, these proviruses were in general clearly recombinant within the p15E-coding sequences. Although the proviral recombination patterns were quite variable, in general the large majority of recombinant proviruses within each tumor appeared structurally identical, indicating that they originate from a common parent. Each tumor contained a unique pattern of provirus integrations; densitometer tracings of the Southern hybridizations indicated that many of the integrated proviruses were present at one copy per cell, suggesting that the tumors derive from a single cell which contained multiple integrated copies of a unique recombinant virus structurally similar to the mink cell focus-inducing viruses.     

Novel host range and cytopathic variant of ecotropic Friend murine leukemia virus

A variant ecotropic Friend murine leukemia virus, F-S MLV, is capable of inducing the formation of large multinucleated syncytia in Mus dunni cells. This cytopathicity resembles that of Spl574 MLV, a novel variant recently isolated from the spleen of a Mus spicilegus mouse neonatally inoculated with Moloney MLV. F-S MLV is an N-tropic Friend MLV that also has the unusual ability to infect hamster cells, which are normally resistant to mouse ecotropic MLVs. Syncytium induction by both F-S MLV and Spl574 is accompanied by the accumulation of large amounts of unintegrated viral DNA, a hallmark of pathogenic retroviruses, but not previously reported for mouse ecotropic gammaretroviruses. Sequencing and site-specific mutagenesis determined that the syncytium-inducing phenotype of F-S MLV can be attributed to a single amino acid substitution (S84A) in the VRA region of the viral env gene. This site corresponds to that of the single substitution previously shown to be responsible for the cytopathicity of Spl574, S82F. The S84A substitution in F-S MLV also contributes to the ability of this virus to infect hamster cells, but Spl574 MLV is unable to infect hamster cells. Because this serine residue is one of the critical amino acids that form the CAT-1 receptor binding site, and because M. dunni and hamster cells have variant CAT-1 receptors, these results suggest that syncytium formation as well as altered host range may be a consequence of altered interaction between virus and receptor.     

A Linkage Map of Endogenous Murine Leukemia Proviruses

Thirty endogenous proviruses belonging to the modified polytropic (Mpmv) class of murine leukemia virus (MLV) were identified by proviral-cellular DNA junction fragment segregation in several sets of recombinant inbred mice. Twenty-six Mpmv loci were mapped to chromosomal regions by matching proviral strain distribution patterns to those of previously assigned genes. Like other endogenous nonecotropic MLVs, Mpmv loci were present on several chromosomes in all strains examined. We pooled recombinant inbred strain linkage data from 110 MLV loci and selected marker genes in order to construct a chromosomal linkage map. Every mouse chromosome was found to harbor at least one proviral insertion, and several regions contained multiple integrations. However, the overall distribution of the 110 mapped proviruses did not deviate significantly from a random distribution. Because of their polymorphism in inbred strains of mice, and the ability to score as many as 57 proviruses per strain using only three hybridization probes, the nonecotropic MLVs mapped in common strains of mice offer a significant advantage over older methods (e.g., biochemical or individual restriction fragment polymorphisms) as genetic markers. These endogenous insertion elements should also be useful for assessing strain purity, and for studying the relatedness of common and not-so-common inbred strains.     

Characterization of endogenous and recombinant proviral elements of a highly tumorigenic AKR cell line

As an approach to evaluating the contribution of classes of endogenous viral sequences to leukemogenesis, a genomic library was prepared from the highly tumorigenic AKR SL12.3 cell line and screened for env-containing proviruses. An extensive battery of virus-derived probes and specific oligonucleotide probes were used to segregate 83 positive clones into related groups. The nonecotropic endogenous retroviruses were identified as members of the polytropic, modified polytropic, or xenotropic groups. At least three unique xenotropic proviruses were detected that differed from the published xenotropic sequence within a variable region of the 5' portion of env. Changes among the xenotropic proviruses included relative insertions and/or deletions that maintain an open reading frame and hence the potential to encode viable envelope gene products. Several recombinant viruses were also detected. Recombination was not random and primarily involved the formation of mink cell focus-inducing class I retroviruses via recombination between polytropic elements and ecotropic virus. One other recombinant was detected which contained ecotropic virus sequences in the 5' region encoding p15 of an otherwise xenotropic provirus. An interesting observation was the finding that certain clones contained more than one provirus within the average 20-kb cloned insert. This would not be expected if integration were totally random. The de novo recombinant proviruses identified here provide a series of potential candidates to be evaluated for their contribution to the tumorigencity of the SL12.3 cell line.     

Polymorphisms of the Cell Surface Receptor Control Mouse Susceptibilities to Xenotropic and Polytropic Leukemia Viruses

The differential susceptibilities of mouse strains to xenotropic and polytropic murine leukemia viruses (X-MLVs and P-MLVs, respectively) are poorly understood but may involve multiple mechanisms. Recent evidence has demonstrated that these viruses use a common cell surface receptor (the X-receptor) for infection of human cells. We describe the properties of X-receptor cDNAs with distinct sequences cloned from five laboratory and wild strains of mice and from hamsters and minks. Expression of these cDNAs in resistant cells conferred susceptibilities to the same viruses that naturally infect the animals from which the cDNAs were derived. Thus, a laboratory mouse (NIH Swiss) X-receptor conferred susceptibility to P-MLVs but not to X-MLVs, whereas those from humans, minks, and several wild mice (Mus dunni, SC-1 cells, and Mus spretus) mediated infections by both X-MLVs and P-MLVs. In contrast, X-receptors from the resistant mouse strain Mus castaneus and from hamsters were inactive as viral receptors. These results suggest that X-receptor polymorphisms are a primary cause of resistances of mice to members of the X-MLV/P-MLV family of retroviruses and are responsible for the xenotropism of X-MLVs in laboratory mice. By site-directed mutagenesis, we substituted sequences between the X-receptors of M. dunni and NIH Swiss mice. The NIH Swiss protein contains two key differences (K500E in presumptive extracellular loop 3 [ECL 3] and a T582 deletion in ECL 4) that are both required to block X-MLV infections. Accordingly, a single inverse mutation in the NIH Swiss protein conferred X-MLV susceptibility. Furthermore, expression of an X-MLV envelope glycoprotein in Chinese hamster ovary cells interfered efficiently with X-MLV and P-MLV infections mediated by X-receptors that contained K500 and/or T582 but had no effect on P-MLV infections mediated by X-receptors that lacked these amino acids. In contrast, moderate expression of a P-MLV (MCF247) envelope glycoprotein did not cause substantial interference, suggesting that X-MLV and P-MLV glycoproteins interfere nonreciprocally with X-receptor-mediated infections. We conclude that P-MLVs have become adapted to utilize X-receptors that lack K500 and T582. A penalty for this adaptation is a reduced ability to interfere with superinfection. Because failure of interference is a hallmark of several exceptionally pathogenic retroviruses, we propose that it contributes to P-MLV-induced diseases.     

Polymorphism of murine endogenous proviruses revealed by using virus class-specific oligonucleotide probes

Inbred mice contain three classes of endogenous nonecotropic murine leukemia virus-related sequences, namely xenotropic, polytropic, and modified polytropic proviruses. Oligonucleotide probes specific for the three different classes were prepared and used to examine the diversity of endogenous sequences present in eight different strains of mice: HRS/J, BALB/cJ, A/J, AKR/J, C57BL/6J, DBA/2J, C57L/J, and C3H/HeJ. A high degree of polymorphism was observed. Overall, the strains showed between 17% (A/J and HRS/J) and 65% (C57BL/6J and C57L/J) shared proviruses, and only four proviruses were present in all eight strains. The similarity among the strains is due in part to the few proviruses present in all of the strains but also represents the independent assortment of a limited set of proviruses. These oligonucleotides provide a basis for determining the stability, distribution, and mutagenic potential of nonecotropic proviruses within the mouse genome.     

Structure and Distribution of Endogenous Nonecotropic Murine Leukemia Viruses in Wild Mice

Virtually all of our present understanding of endogenous murine leukemia viruses (MLVs) is based on studies with inbred mice. To develop a better understanding of the interaction between endogenous retroviruses and their hosts, we have carried out a systematic investigation of endogenous nonecotropic MLVs in wild mice. Species studied included four major subspecies of Mus musculus (M. m. castaneus, M. m. musculus, M. m. molossinus, and M. m. domesticus) as well as four common inbred laboratory strains (AKR/J, HRS/J, C3H/HeJ, and C57BL/6J). We determined the detailed distribution of nonecotropic proviruses in the mice by using both env- and long terminal repeat (LTR)-derived oligonucleotide probes specific for the three different groups of endogenous MLVs. The analysis indicated that proviruses that react with all of the specific probes are present in most wild mouse DNAs tested, in numbers varying from 1 or 2 to more than 50. Although in common inbred laboratory strains the linkage of group-specific sequences in env and the LTR of the proviruses is strict, proviruses which combine env and the LTR sequences from different groups were commonly observed in the wild-mouse subspecies. The “recombinant” nonecotropic proviruses in the mouse genomes were amplified by PCR, and their genetic and recombinant natures were determined. These proviruses showed extended genetic variation and provide a valuable probe for study of the evolutionary relationship between MLVs and the murine hosts.