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.     

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.     

Characterization of proviruses cloned from mink cell focus-forming virus-infected cellular DNA

Two proviruses were cloned from EcoRI-digested DNA extracted from mink cells chronically infected with AKR mink cell focus-forming (MCF) 247 murine leukemia virus (MuLV), using a lambda phage host vector system. One cloned MuLV DNA fragment (designated MCF 1) contained sequences extending 6.8 kilobases from an EcoRI restriction site in the 5' long terminal repeat (LTR) to an EcoRI site located in the envelope (env) region and was indistinguishable by restriction endonuclease mapping for 5.1 kilobases (except for the EcoRI site in the LTR) from the 5' end of AKR ecotropic proviral DNA. The DNA segment extending from 5.1 to 6.8 kilobases contained several restriction sites that were not present in the AKR ecotropic provirus. A 0.5-kilobase DNA segment located at the 3' end of MCF 1 DNA contained sequences which hybridized to a xenotropic env-specific DNA probe but not to labeled ecotropic env-specific DNA. This dual character of MCF 1 proviral DNA was also confirmed by analyzing heteroduplex molecules by electron microscopy. The second cloned proviral DNA (designated MCF 2) was a 6.9-kilobase EcoRI DNA fragment which contained LTR sequences at each end and a 2.0-kilobase deletion encompassing most of the env region. The MCF 2 proviral DNA proved to be a useful reagent for detecting LTRs electron microscopically due to the presence of nonoverlapping, terminally located LTR sequences which effected its circularization with DNAs containing homologous LTR sequences. Nucleotide sequence analysis demonstrated the presence of a 104-base-pair direct repeat in the LTR of MCF 2 DNA. In contrast, only a single copy of the reiterated component of the direct repeat was present in MCF 1 DNA.     

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.     

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.     

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

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

Discrete regions of sequence homology between cloned rodent VL30 genetic elements and AKV-related MuLV provirus genomes

Southern blot analyses using reduced stringency hybridization conditions have been employed to search for sequence homologies between rodent VL30 genes and murine leukemia virus (MuLV) proviruses. These constitute two classes of transposon-like elements previously believed to be genetically unrelated. Our results demonstrate that cloned representatives of both ecotropic and xenotropic-like proviruses share discrete regions of sequence homology with VL30 genes of both rat and mouse origin. These regions of homology exist in both 3' and 5' halves of the MuLV genome but do not include extensive portions of the long terminal repeat (LTR) or a 0.4 Kbp segment of the env gene specific for recently acquired ecotropic-type MuLV proviruses. DNA sequencing, however, revealed that the short inverted terminal repeat sequence of MuLV proviral LTRs is almost perfectly conserved at the terminus of an integrated mouse VL30 gene. These results suggest that recombination events with rodent VL30-type sequences occurred during early MuLV evolution. The strong conservation of the inverted terminal repeat sequence may reflect a common integration mechanism for VL30 elements and MuLV proviruses.     

Cas-Br-E murine leukemia virus: sequencing of the paralytogenic region of its genome and derivation of specific probes to study its origin and the structure of its recombinant genomes in leukemic tissues.

The ecotropic Cas-Br-E murine leukemia virus (MuLV) and its molecularly cloned derivative pBR-NE-8 MuLV are capable of inducing hind-limb paralysis and leukemia after inoculation into susceptible mice. T1 oligonucleotide fingerprinting, molecular hybridization, and restriction enzyme analysis previously showed that the env gene of Cas-Br-E MuLV diverged the most from that of other ecotropic MuLVs. To analyze proviruses in leukemic tissues, we derived DNA probes specific to Cas-Br-E sequences: two from the env region and one from the U3 long terminal repeat. With these probes, we found that this virus induced clonal (or oligoclonal) tumors and we documented the presence of typical mink cell focus-forming-type proviruses in leukemic tissues and the possible presence of other recombinant MuLV proviruses. Since the region harboring the determinant of paralysis was mapped within the pol-env region of the virus (L. DesGroseillers, M. Barrette, and P. Jolicoeur, J. Virol. 52:356-363, 1984), we performed the complete nucleotide sequence of this region covering the 3' end of the genome. We compared the deduced amino acid sequences of the pol carboxy-terminal domain and of the env gene products with those of other nonparalytogenic, ecotropic, and mink cell focus-forming MuLVs. This amino acid comparison revealed that this part of the pol gene product and the p15E diverged very little from homologous proteins of other MuLVs. However, the Cas-Br-E gp70 sequence was found to be quite divergent from that of other MuLVs, and the amino acid changes were distributed all along the protein. Therefore, gp70 remains the best candidate for harboring the 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.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.     

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.     

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.     

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.     

Analysis of the env gene of a molecularly cloned and biologically active Moloney mink cell focus-forming proviral DNA.

A biologically active molecular clone of BALB/Moloney mink cell focus-forming (Mo-MCF) proviral DNA has been reconstructed in vitro. It contains the 5' half of BALB/Moloney murine leukemia virus (Mo-MuLV) DNA and the 3' half of BALB/Mo-MCF DNA. The complete nucleotide sequence of the env gene and the 3' long terminal repeat (LTR) of the cloned Mo-MCF DNA has been determined and compared with the sequence of the corresponding region of parental Mo-MuLV DNA. The substitution in the Mo-MCF DNA encompasses 1,159 base pairs, beginning in the carboxyl terminus of the pol gene and extending to the middle of the env gene. The Mo-MCF env gene product is predicted to be 29 amino acids shorter than the parental Mo-MuLV env gene product. The portion of the env gene encoding the p15E peptide is identical in both viral DNAs. There is an additional A residue in the Mo-MCF viral DNA in a region just preceding the 3' LTR. The nucleotide sequence of the 3' LTR of Mo-MCF DNA is similar to that of the 5' LTR of BALB/Mo-MuLV DNA with the exception of two single base substitutions. We conclude that the sequence substitution in the env gene is responsible for the dual-tropic properties of Mo-MCF viruses.     

Genomes of murine leukemia viruses isolated from wild mice.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.     

Kinetics of expression of infectious ecotropic, xenotropic, and mink cell focus-forming murine leukemia virus after 5-iododeoxyuridine induction of cells from high- and low-leukemia mouse strains

Endogenous murine leukemia virus (MuLV) was induced with 5-iododeoxyuridine (IdUrd) from the high-leukemia mouse strain AKR and from two low-leukemia strains, C3H/He and BALB/c. A virus-free cell line from strain AKR readily gave rise to infectious, XC-positive MuLV upon treatment with IdUrd, whereas cells from strains C3H/He and BALB/c produced replication-deficient, XC-negative MuLV. IdUrd-induced cells also produced xenotropic and mink cell focus-forming MuLV. Xenotropic virus emerged at a higher titer from both AKR and BALB/c cells during two discrete time intervals, first at day 3 after induction and a second time during spread of the induced ecotropic MuLV. Xenotropic and mink cell focus-forming MuLVs were also produced by IdUrd-induced C3H/He cells but required another round of infection in Sc-1 cells for detection. The in vitro infectivity of endogenous ecotropic MuLV isolated by IdUrd induction from C3H/He cells correlated with pathogenicity in the Fv-1-compatible, leukemia-negative mouse strain NFS/N. Thus, the virulence of endogenous ecotropic MuLV may be an important factor in determining the leukemia incidence in these inbred strains of mice.     

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.     

Molecular cloning of viral DNA from leukemogenic Gross passage A murine leukemia virus and nucleotide sequence of its long terminal repeat.

The viral DNA genome of the leukemogenic Gross passage A virus was cloned in phage Charon 21A as an infectious molecule. The virus recovered by transfection with this infectious DNA was ecotropic, N-tropic, fibrotropic, and XC+. It was leukemogenic when reinjected into newborn SIM mice, indicating that ecotropic murine leukemia virus (MuLV) from an AKR mouse thymoma can harbor leukemogenic sequences. Its restriction map was similar to that of nonleukemogenic AKR MuLV, its putative parent, but differed at the 3' end and in the long terminal repeat (LTR). The nucleotide sequence of the Gross A virus LTR was identical to the AKR MuLV LTR sequence (Van Beveren et al., J. Virol. 41:542-556, 1982) in U5, R, and part of U3. All differences between both LTRs were found in U3. Only one copy of the U3 tandem direct repeat was conserved in the Gross A virus LTR, and it was rearranged by the insertion of a 36-base-pair sequence and by five point mutations. Only one additional point mutation common to several oncogenic MuLVs was present in U3. These structural changes in the U3 LTR and at the 3' end of the genome may be related to the leukemogenicity of this virus.