Nucleotide sequence analysis establishes the role of endogenous murine leukemia virus DNA segments in formation of recombinant mink cell focus-forming murine leukemia viruses.

The sequence of 363 nucleotides near the 3' end of the pol gene and 564 nucleotides from the 5' terminus of the env gene in an endogenous murine leukemia viral (MuLV) DNA segment, cloned from AKR/J mouse DNA and designated as A-12, was obtained. For comparison, the nucleotide sequence in an analogous portion of AKR mink cell focus-forming (MCF) 247 MuLV provirus was also determined. Sequence features unique to MCF247 MuLV DNA in the 3' pol and 5' env regions were identified by comparison with nucleotide sequences in analogous regions of NFS -Th-1 xenotropic and AKR ecotropic MuLV proviruses. These included (i) an insertion of 12 base pairs encoding four amino acids located 60 base pairs from the 3' terminus of the pol gene and immediately preceding the env gene, (ii) the deletion of 12 base pairs (encoding four amino acids) and the insertion of 3 base pairs (encoding one amino acid) in the 5' portion of the env gene, and (iii) single base substitutions resulting in 2 MCF247 -specific amino acids in the 3' pol and 23 in the 5' env regions. Nucleotide sequence comparison involving the 3' pol and 5' env regions of AKR MCF247 , NFS xenotropic, and AKR ecotropic MuLV proviruses with the cloned endogenous MuLV DNA indicated that MCF247 proviral DNA sequences were conserved in the cloned endogenous MuLV proviral segment. In fact, total nucleotide sequence identity existed between the endogenous MuLV DNA and the MCF247 MuLV provirus in the 3' portion of the pol gene. In the 5' env region, only 4 of 564 nucleotides were different, resulting in three amino acid changes between AKR MCF247 MuLV DNA and the endogenous MuLV DNA present in clone A-12. In addition, nucleotide sequence comparison indicated that Moloney-and Friend-MCF MuLVs were also highly related in the 3' pol and 5' env regions to the cloned endogenous MuLV DNA. These results establish the role of endogenous MuLV DNA segments in generation of recombinant MCF viruses.     

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.     

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.     

Molecular characterization of a neuropathogenic and nonerythroleukemogenic variant of Friend murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a replication-competent, ecotropic type C retrovirus that was isolated after passage of the Friend virus complex through F344 rats. Unlike viruses in the Friend virus complex, it does not cause erythroleukemia but causes a rapidly progressive hind limb paralysis when injected into newborn rats and mice. We have isolated an infectious DNA clone (clone 3d) of this virus which causes neurological disease in animals as efficiently as parental PVC-211 MuLV. The restriction map of clone 3d is very similar to that of the nonneuropathogenic, erythroleukemogenic Friend murine leukemia virus (F-MuLV), suggesting that PVC-211 MuLV is a variant of F-MuLV and that no major structural alteration was involved in its derivation. Studies with chimeric viruses between PVC-211 MuLV clone 3d and wild-type F-MuLV clone 57 indicate that at least one determinant for neuropathogenicity resides in the 2.1-kb XbaI-ClaI fragment containing the gp70 coding region of PVC-211 MuLV. Compared with nonneuropathogenic ecotropic MuLVs, the env gene of PVC-211 MuLV encodes four unique amino acids in the gp70 protein. Nucleotide sequence analysis also revealed a deletion in the U3 region of the long terminal repeat (LTR) of PVC-211 MuLV clone 3d compared with F-MuLV clone 57. In contrast to the env gene of PVC-211 MuLV, particular sequences within the U3 region of the viral LTR do not appear to be required for neuropathogenicity. However, the changes in the LTR of PVC-211 MuLV may be responsible for the failure of this virus to cause erythroleukemia, because chimeric viruses containing the U3 region of F-MuLV clone 57 were erythroleukemogenic whereas those with the U3 of PVC-211 MuLV clone 3d were not.     

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.     

Physical mapping of the paralysis-inducing determinant of a wild mouse ecotropic neurotropic retrovirus.

We have recently shown that a molecularly cloned ecotropic retrovirus, initially isolated from the brain of a paralyzed wild mouse, retained the ability to induce hind limb paralysis when inoculated into susceptible mice (Jolicoeur et al., J. Virol. 45:1159-1163, 1983). To map the viral DNA sequences encoding the determinant of paralysis, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from this neurotropic murine leukemia virus (MuLV) and from nonneurotropic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered after microinjection of NIH 3T3 cells with these recombinant DNAs, were inoculated into newborn SIM.S and SWR/J mice to test the paralysis-inducing potential. We found that the 3.9-kilobase-pair SalI-ClaI fragment of the neurotropic MuLV comprising the 3' end of pol and all env sequences was sufficient to confer the paralysis-inducing potential to chimeric viruses. Therefore, this region of the neurotropic MuLV genome most likely harbors the primary determinant of paralysis.     

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.     

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.     

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.     

Specific hybridization probes demonstrate fewer xenotropic than mink cell focus-forming murine leukemia virus env-related sequences in DNAs from inbred laboratory mice.

We have derived hybridization probes from analogous 100-base-pair segments located within the N-terminal region of gp70 coding sequences which differentiate xenotropic from mink cell focus-forming (MCF)-related murine leukemia virus (MuLV) DNAs. The MCF probe annealed to the integrated proviruses of all six MCF MuLV isolates tested; the xenotropic probe hybridized to the DNAs of all four xenotropic proviral isolates examined. No cross-hybridization was observed, and neither probe reacted with the env segments of amphotropic or ecotropic MuLV DNAs. Southern blot analysis of HindIII- or EcoRI-digested genomic DNAs from a variety of inbred laboratory mice demonstrated the presence of more MCF- than xenotropic MuLV-related segments in every strain tested.     

New class of leukemogenic ecotropic recombinant murine leukemia virus isolated from radiation-induced thymomas of C57BL/6 mice

We previously reported the establishment of several lymphoid cell lines from X-ray-induced thymomas of C57BL/Ka mice, and all, except one, produce retroviruses (P. Sankar-Mistry and P. Jolicoeur, J. Virol.35:270-275, 1980). Biological characterization of five of these new primary radiation leukemia viruses (RadLVs) indicated that they had a B-tropic, fibrotropic, and ecotropic host range and were leukemogenic when reinjected into C57BL/Ka newborn mice. The leukemogenic potential of one isolate (G(6)T(2)) was further assessed and shown to be retained after prolonged passaging on fibroblasts in vitro. Restriction endonuclease analysis of the DNA of four of our new RadLV isolates (G(6)T(2), Ti-7, Ti-8, and Ti-9) revealed that G(6)T(2) and Ti-7 murine leukemia virus (MuLV) genomes had identical restriction maps, whereas Ti-8 and Ti-9 genomes were different from each other and from the G(6)T(2) and Ti-7 genomes. The physical maps of these genomes were similar to that of known ecotropic MuLV genomes (including the C57BL/Ka endogenous ecotropic MuLV) within their long terminal repeats, env, the right portion of pol, and the left portion of gag. However, a region covering the end of gag and the beginning of pol was different and showed several similarities with xenotropic MuLV genomes of BALB/c, AKR, and C58 mice previously mapped. Our results suggest that these primary RadLV genomes are recombinants between the parental ecotropic MuLV genome and a nonecotropic (xenotropic) sequence. This nonecotropic gag-pol region might be important in conferring the leukemogenic potential to these isolates. Therefore, these RadLVs appear to form a new class of leukemogenic recombinant MuLVs recovered from leukemic tissues of mice. They appear to be distinct from the recombinant AKR mink cell focus-inducing MuLVs which have a dual-tropic host range and harbor xenotropic env sequences. To further study the leukemogenic potential of these RadLVs, the genome of one of them (G(6)T(2)) was cloned in Charon 21A as an infectious molecule.     

Molecular cloning of infectious viral DNA from ecotropic neurotropic wild mouse retrovirus.

Among a mixture of amphotropic and ecotropic murine leukemia viruses (MuLVs) isolated from paralyzed wild mice, only N-tropic ecotropic MuLV, cloned by cell culture techniques, has been shown to induce paralysis after reinjection into susceptible mice (M. B. Gardner, Curr. Top. Microbiol. Immunol. 79:215-239, 1978). The viral DNA genome of one of these neurotropic MuLVs (Cas-Br-E) has been cloned in Charon 21A at the SalI site. One clone, designated NE-8, was studied in more detail. A restriction endonuclease map of this cloned DNA was derived. Cloned viral DNA microinjected into NIH 3T3 cells produced infectious MuLV which was characterized as XC+, ecotropic, and N-tropic. The virus that was recovered after the microinjection of NE-8 DNA was also injected into susceptible SIM.S and NIH Swiss mice and was found to induce lower limb paralysis in these animals. These results make it highly unlikely that other agents (which might have escaped detection and separation from ecotropic MuLV by the techniques previously used) play a role in the etiology of this disease and clearly indicate that the ecotropic MuLV genome harbors sequences responsible for this paralysis. The availability of this clone DNA would now allow us to map these sequences on the genome.     

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.     

Both the changes of six amino acids and the C-terminal truncation caused by a one-base insertion in the defective env gene of Friend spleen focus-forming virus significantly affect the pathogenic activity of the encoded leukemogenic membrane glycoprotein (gp55).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in mice and encodes in its defective env gene an Env-like membrane glycoprotein (gp55). The F-SFFV env gene has three characteristic structures compared with that of ecotropic murine leukemia viruses (MuLVs): substitution by the polytropic MuLV env sequence, a 585-bp deletion, and a 1-bp insertion. All of these characteristic structures are essential for the leukemogenic potential of gp55 of polycythemia-inducing isolates of F-SFFV (F-SFFVp). The 1-bp insertion causes changes of six amino acids and truncation by 34 amino acids at the C terminus. In this study, we constructed 12 mutant F-SFFV genomes starting from the wild-type F-SFFVp and examined the effect of the C-terminal truncation and the six altered amino acids on the pathogenic activity of gp55. The results indicated that at least 18 to 24 amino acids must be deleted from the C terminus for the env product to be pathogenically active. We also found that the six altered amino acids contributed significantly to the pathogenic activity of gp55. Analyses of the cellular processing of these mutant gp55s supported a correlation between the pathogenic activity of gp55 and its efficiency in overall cellular processing.     

Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences.

We sequenced the envelope (env) gene and 3' long terminal repeat of a Friend mink cell focus-inducing virus (F-MCFV). We also sequenced the gp70 coding regions for two cDNA clones of another F-MCFV. The deduced amino acid sequence of the env gene products of both F-MCFVs were compared to the corresponding sequences of other MCFVs and of ecotropic viruses. The env polypeptides of the different viruses showed long stretches of homology in the carboxy-terminal half of gp70 and in p15env ("constant region"). The amino-terminal half of gp70 was very similar in all MCFVs, but showed extensive variations relative to the ecotropic viruses ("differential region"). This differential region in all MCFVs is of endogeneous origin. We show evidence that this region carries determinants for ecotropic or polytropic host range. No indication could be found that the env gene products determine the histological type of disease caused by particular MCFVs. When the long terminal repeats of F-MCFV and Friend murine leukemia virus were compared with those of other viruses causing either lymphatic leukemia or erythroleukemia, several nucleotides were localized which might determine the histological type of disease caused by these viruses.     

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.     

Comparison of endogenous murine leukemia virus proviral organization and RNA expression in 3-methylcholanthrene-induced and spontaneous thymic lymphomas in RF and AKR mice.

3-Methylcholanthrene-induced T-cell thymic lymphomas in RF mice were examined for involvement of murine leukemia virus (MuLV)-related sequences in leukemogenesis. Both the expression of MuLV-related RNA species and the organization of endogenous MuLV proviral DNA were analyzed. Of 27 primary tumors examined, only 5 exhibited elevated MuLV-related RNA species homologous to xenotropic specific env DNA. None of these RNA species hybridized with ecotropic p15E DNA sequences. Only two of these five tumors contained MuLV-like RNA species that hybridized with ecotropic MuLV long terminal repeat sequences, despite the probe's ability to detect both ecotropic MuLV and mink cell focus-inducing viral RNA. No muLV resembling mink cell focus-inducing virus whose expression could be correlated with lymphomagenesis was detected in either preleukemic thymocytes, primary 3-methylcholanthrene-induced thymic tumors, tumors passaged in vivo, or cell lines derived from tumors. Restriction endonuclease analysis of DNA from both primary tumors and cell lines failed to reveal either proviral DNA with recombinant env genes or rearrangement of endogenous MuLV proviruses. Therefore, chemically induced lymphomagenesis in RF mice appears different from the spontaneous lymphomagenic process in AKR mice with respect to the involvement of endogenous MuLV sequences.     

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.     

Mus cervicolor murine leukemia virus isolate M813 belongs to a unique receptor interference group

Murine leukemia virus (MuLV) M813 was originally isolated from the Southeast Asian rodent Mus cervicolor. As with the ecotropic MuLVs derived from Mus musculus, its host range is limited to rodent cells. Earlier studies have mapped its receptor to chromosome 2, but it has not been established whether M813 shares a common receptor with any other MuLVs. In this study, we have performed interference assays with M813 and viruses from four interference groups of MuLV. The infection efficiency of M813 was not compromised in cells expressing any one of the other MuLVs, demonstrating that M813 must use a distinct receptor for cell entry. The entire M813 env coding region was molecularly cloned. Sequence analysis revealed high similarity with other MuLVs but with a unique receptor-binding domain. Substitution of M813 env sequences in Moloney MuLV resulted in a replication-competent virus with a host range and interference profile similar to those of the biological clone M813. M813 thus defines a novel receptor interference group of type C MuLVs.     

The high leukemogenic potential of Gross passage A murine leukemia virus maps in the region of the genome corresponding to the long terminal repeat and to the 3' end of env.

The Gross passage A murine leukemia virus (MuLV) is a highly leukemogenic, ecotropic fibrotropic retrovirus. Its genome is similar to that of other nonleukemogenic ecotropic fibrotropic MuLVs but differs at the 3' end and in the long terminal repeat. To determine whether these modifications were related to its leukemogenic potential, we constructed a viral DNA recombinant in vitro with cloned infectious DNA from this highly leukemogenic Gross passage A MuLV and from a weakly leukemogenic endogenous BALB/c B-tropic MuLV. Infectious viruses, recovered after microinjection of murine cells with recombinant DNA, were injected into newborn mice. We show here that the Gross passage A 1.35-kilobase-pair KpnI fragment (harboring part of gp70, all of p15E, and the long terminal repeat) is sufficient to confer a high leukemogenic potential to this recombinant.