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.     

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.     

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.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag-pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

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.     

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.     

Inhibition of infectious murine leukemia virus production by Fv-4 env gene products exerting dominant negative effect on viral envelope glycoprotein

Fv-4 is a mouse gene that confers resistance against ecotropic murine leukemia virus (MLV) infection on mice. While receptor interference by the Fv-4 env gene product, Fv-4 Env, that can bind to the ecotropic MLV receptor has been shown to play an important role in the resistance, other mechanisms have also been suggested because it confers extremely efficient, complete resistance in vivo. Here, we have examined the effect of Fv-4 Env on infectious MLV production. Infectious MLV titers in supernatants obtained after transfection with a Friend MLV (FMLV) Env-expressing plasmid from MLV gag–pol producer cells harboring a retroviral vector were largely reduced by coexpression of Fv-4 Env. Syncytia formation mediated by R-peptide-deleted FMLV Env in NIH 3T3 cells was impaired by Fv-4 Env coexpression. Similarly, Fv-4 Env inhibited infectious amphotropic MLV production and syncytia formation mediated by R-peptide-deleted amphotropic MLV Env. Immunoprecipitation analysis revealed interaction of Fv-4 Env with amphotropic MLV Env as well as FMLV Env. These results indicate that Fv-4 Env inhibits infectious ecotropic and amphotropic MLV production by exerting dominant negative effect on MLV Env, suggesting contribution of this inhibitory effect to the resistance against ecotropic MLV infection in Fv-4-expressing mice.     

A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses.

An epitope common to all classes of murine leukemia viruses (MuLVs) was detected by reactivity of MuLVs with a rat monoclonal antibody (MAb) termed 83A25. The antibody is of the immunoglobulin G2a isotype and was derived after fusion of NS-1 myeloma cells with spleen cells from a Fischer rat immunized with a Friend polytropic MuLV. The antibody reacted with nearly all members of the ecotropic, polytropic, xenotropic, and amphotropic classes of MuLVs. Unreactive viruses were limited to the Friend ecotropic MuLV, Rauscher MuLV, and certain recombinant derivatives of Friend ecotropic MuLV. The presence of an epitope common to nearly all MuLVs facilitated a direct quantitative focal immunofluorescence assay for MuLVs, including the amphotropic MuLVs for which no direct assay has been previously available. Previously described MAbs which react with all classes of MuLVs have been limited to those which react with virion core or transmembrane proteins. In contrast, protein immunoblot and immunoprecipitation analyses established that the epitope reactive with MAb 83A25 resides in the envelope glycoproteins of the viruses. Structural comparisons of reactive and nonreactive Friend polytropic viruses localized the epitope near the carboxyl terminus of the glycoprotein. The epitope served as a target for neutralization of all classes of MuLV with MAb 83A25. The efficiency of neutralization varied with different MuLV isolates but did not correlate with MuLV interference groups.     

Molecular characterization of the Akvr-1 restriction gene: a defective endogenous retrovirus-borne gene identical to Fv-4r.

A dominant restriction allele, Akvr-1r, from California wild mice (Mus musculus domesticus) confers resistance to exogenous ecotropic murine leukemia virus (MuLV) infection. The presence of an ecotropic MuLV envelope-related glycoprotein in uninfected virus-resistant cells suggests that viral interference is a possible mechanism for this resistance. We molecularly cloned the ecotropic MuLV envelope-related sequence from the genomic DNA of a wild mouse homozygous for the Akvr-1r locus. The cloned provirus was defective and contained a C-terminal end of the pol gene, a complete envelope gene, and a 3' long terminal repeat. The presence of this provirus was directly correlated with Akvr-1r-mediated virus resistance in cell cultures and hybrid mice. The Akvr-1r provirus restriction map and partial DNA sequence were identical to those of the Fv-4r allele, an ecotropic MuLV resistance locus from Japanese feral mice (M. musculus molossinus), which was previously shown to be allelic with the Akvr-1r gene. The 3' host flanking sequences of Fv-4r and Akvr-1r also had identical restriction maps. These findings indicate that Akvr-1r and Fv-4r are the same gene. It was probably acquired by interbreeding of these feral species in recent times. Conservation of this locus might be favored by the useful function that it performs in protection against ecotropic MuLV infection endemic in both populations of wild mice.     

Mechanism of selection of class II recombinant murine leukemia viruses in the highly leukemic strain CWD

The development of spontaneous lymphomas in CWD mice is associated with the expression of endogenous ecotropic murine leukemia viruses (MuLV) and the formation of recombinant viruses. However, the pattern of substitution of nonecotropic sequences within the envelope genes of the CWD class II recombinant viruses differs from that seen in class I recombinant MuLVs of AKR, C58, and HRS mice. To determine how CWD host genes might influence the envelope gene structure of the recombinant viruses, we characterized the responses of these mice to two different types of exogenous MuLVs. Neonatal mice injected the HRS class I recombinant PTV-1 became infected and developed T-cell lymphomas more rapidly than controls did. The inoculation of CWD mice with the leukemogenic AKR ecotropic virus SL3-3 led to the formation of recombinant MuLVs with a novel genetic structure and class II-like envelope genes, although SL3-3 generates class I recombinants in other strains. These results suggest that the absence of class I recombinant MuLVs in CWD mice is not related to the restriction of the replication or oncogenicity of class I viruses or to the absence of an appropriate ecotropic virus that can generate class I recombinants. More likely, the genes of CWD mice that direct the formation or selection of class II recombinant viruses affect the process of recombination between the ecotropic and nonecotropic envelope gene sequences.     

Functional characterization of the N termini of murine leukemia virus envelope proteins

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.     

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.     

Phenotypes of murine leukemia virus-induced tumors: influence of 3'' viral coding sequences.

Murine leukemia viruses (MuLVs) induce leukemias and lymphomas in mice. We have used fluorescence-activated cell sorter analysis to determine the hematopoietic phenotypes of tumor cells induced by a number of MuLVs. Tumor cells induced by ecotropic Moloney, amphotropic 4070A, and 10A1 MuLVs and by two chimeric MuLVs, Mo(4070A) and Mo(10A1), were examined with antibodies to 13 lineage-specific cell surface markers found on myeloid cell, T-cell, and B-cell lineages. The chimeric Mo(4070A) and Mo(10A1) MuLVs, consisting of Moloney MuLV with the carboxy half of the Pol region and nearly all of the Env region of 4070A and 10A1, respectively, were constructed to examine the possible influence of these sequences on Moloney MuLV-induced tumor cell phenotypes. In some instances, these phenotypic analyses were supplemented by Southern blot analysis for lymphoid cell-specific genomic DNA rearrangements at the immunoglobulin heavy-chain, the T-cell receptor gamma, and the T-cell receptor beta loci. The results of our analysis showed that Moloney MuLV, 4070A, Mo(4070A), and Mo(10A1) induced mostly T-cell tumors. Moloney MuLV and Mo(4070A) induced a wide variety of T-cell phenotypes, ranging from immature to mature phenotypes, while 4070A induced mostly prothymocyte and double-negative (CD4- CD8-) T-cell tumors. The tumor phenotypes obtained with 10A1 and Mo(10A1) were each less variable than those obtained with the other MuLVs tested. 10A1 uniformly induced a tumor consisting of lineage marker-negative cells that lack lymphoid cell-specific DNA rearrangements and histologically appear to be early undifferentiated erythroid cell-like precursors. The Mo(10A1) chimera consistently induced an intermediate T-cell tumor. The chimeric constructions demonstrated that while 4070A 3' pol and env sequences apparently did not influence the observed tumor cell phenotypes, the 10A1 half of pol and env had a strong effect on the phenotypes induced by Mo(10A1) that resulted in a phenotypic consistency not seen with other viruses. This result implicates 10A1 env in an active role in the tumorigenic process.     

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.     

Influence of murine leukemia proviral integrations on development of N-methyl-N-nitrosourea-induced thymic lymphomas in AKR mice.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.     

Transgenic Fv-4 mice resistant to Friend virus.

Fv-4 is a mouse gene that confers resistance to infection with ecotropic retroviruses. A candidate Fv-4 gene was cloned previously and found to resemble the 3' half of a murine leukemia virus (MuLV). To study the effect of this gene in vivo, we generated two transgenic mouse strains carrying the Fv-4 env gene under control of its presumed natural promoter, a cellular sequence unrelated to retroviruses. Transgenic progeny expressed a 3-kb Fv-4 env RNA in all of the organs and tissues examined, as well as an Fv-4 envelope antigen on the surface of thymocytes and spleen cells, similar to mice carrying the natural Fv-4 gene. One of the two transgenic strains (designated Fv4-2) expressed three to nine times as much transgene RNA and protein as the other strain (Fv4-11). When challenged with a Friend virus complex containing up to 10(4) XC PFU of Friend MuLV, Fv4-2 mice were completely resistant to development of splenomegaly and had no detectable ecotropic virus in the spleen or blood, confirming that the cloned Fv-4 gene is responsible for resistance to ecotropic MuLV in vivo. In contrast, Fv4-11 mice were only partially resistant, developing viremia and splenomegaly at the highest inoculum dose but recovering from viremia several weeks after inoculation with 10-fold less virus. The phenotype of recovery from viremia in Fv4-11 mice was unexpected and suggests that low levels of expression of the Fv-4 gene enhance the effectiveness of the immune response.     

Characterization of a molecularly cloned retroviral sequence associated with Fv-4 resistance.

The murine leukemia virus (MuLV) sequence associated with the resistance allele of the Fv-4 gene (Fv-4r) was molecularly cloned from genomic DNA of uninfected mice carrying this allele. The 5.2-kilobase cloned EcoRI DNA fragment (pFv4) was shown by nucleotide sequencing to contain 3.4 kilobases of a colinear MuLV-related proviral sequence which began in the C-terminal end of the pol region and extended through the env region and the 3' long terminal repeat. Cellular sequences flanked the 3' as well as the 5' ends of the truncated MuLV sequence. Alignment of the N-terminal half of the pFv4 env sequence with ecotropic, mink cell focus-forming, and xenotropic MuLV env sequences established the relatedness of pFv4 and ecotropic MuLV env sequences. A subcloned 700-base pair segment (pFv4env) from the 5' env region of pFv4 was used as an Fv-4-specific probe; it hybridized specifically to the Fv-4r-associated proviral sequence but not to endogenous ecotropic MuLV proviral DNA under high stringency. All Fv-4-resistant mice contained the same retroviral segment associated with the same flanking cellular DNA. Expression of Fv-4r-specific mRNA was demonstrated in the spleens of Fv-4r mice but not Fv-4s mice, supporting the previously proposed resistance model based on interference.     

Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein.

In the murine leukemia viruses (MuLVs), the Env complex is initially cleaved by a cellular protease into gp70SU and pre15ETM. After the virus particle is released from the cell, the C-terminal 16 residues are removed from the cytoplasmic domain of pre15E by the viral protease, yielding the mature p15ETM and p2E. We have investigated the function of this cleavage by generating a Moloney MuLV mutant, termed p2E-, in which the Env coding region terminates at the cleavage site. This mutant synthesizes only the truncated, mature form of TM rather than its extended precursor. When cells expressing this truncated Env protein are cocultivated with NIH 3T3 cells, they induce rapid cell-cell fusion. Thus, the truncated form, which is normally found in virions but not in virus-producing cells, is capable of causing membrane fusion. We conclude that the 16-residue p2E tail inhibits this activity of Env until the virus has left the cell. p2E- virions were found to be infectious, though with a lower specific infectivity than that of the wild type, showing that p2E does not play an essential role in the process of infection. Fusion was also observed with a chimeric p2E- virus in which gp70SU and nearly all of p15ETM are derived from amphotropic, rather than Moloney, MuLV. In a second mutant, an amino acid at the cleavage site was changed. The pre15E protein in this mutant is not cleaved. While the mutant Env complex is incorporated into virions, these particles have a very low specific infectivity. This result suggests that the cleavage event is essential for infectivity, in agreement with the idea that removal of p2E activates the membrane fusion capability of the Env complex.     

Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus.

Certain mouse strains, such as AKR and C58, which possess N-tropic, ecotropic murine leukemia virus (MuLV) proviruses and are homozygous at the Fv-1n locus are specifically susceptible to paralytic infection (age-dependent poliomyelitis [ADPM]) by lactate dehydrogenase-elevating virus (LDV). Our results provide an explanation for this genetic linkage and directly prove that ecotropic MuLV infection of spinal cord cells is responsible for rendering anterior horn neurons susceptible to cytocidal LDV infection, which is the cause of the paralytic disease. Northern (RNA) blot hybridization of total tissue RNA and in situ hybridization of tissue sections demonstrated that only mice harboring central nervous system (CNS) cells that expressed ecotropic MuLV were susceptible to ADPM. Our evidence indicates that the ecotropic MuLV RNA is transcribed in CNS cells from ecotropic MuLV proviruses that have been acquired by infection with exogenous ecotropic MuLV, probably during embryogenesis, the time when germ line proviruses in AKR and C58 mice first become activated. In young mice, MuLV RNA-containing cells were found exclusively in white-matter tracts and therefore were glial cells. An increase in the ADPM susceptibility of the mice with advancing age correlated with the presence of an increased number of ecotropic MuLV RNA-containing cells in the spinal cords which, in turn, correlated with an increase in the number of unmethylated proviruses in the DNA extracted from spinal cords. Studies with AKXD recombinant inbred strains showed that possession of a single replication-competent ecotropic MuLV provirus (emv-11) by Fv-1n/n mice was sufficient to result in ecotropic MuLV infection of CNS cells and ADPM susceptibility. In contrast, no ecotropic MuLV RNA-positive cells were present in the CNSs of mice carrying defective ecotropic MuLV proviruses (emv-3 or emv-13) or in which ecotropic MuLV replication was blocked by the Fv-1n/b or Fv-1b/b phenotype. Such mice were resistant to paralytic LDV infection. In utero infection of CE/J mice, which are devoid of any endogenous ecotropic MuLVs, with the infectious clone of emv-11 (AKR-623) resulted in the infection of CNS cells, and the mice became ADPM susceptible, whereas littermates that had not become infected with ecotropic MuLV remained ADPM resistant.