Mapping the viral sequences conferring leukemogenicity and disease specificity in Moloney and amphotropic murine leukemia viruses.

The Moloney murine leukemia virus (MuLV) is a highly leukemogenic virus. To map the leukemogenic potential of Moloney MuLV, we constructed chimeric viral DNA genomes in vitro between parental cloned infectious viral DNA from Moloney and amphotropic 4070-A MuLVs. Infectious chimeric MuLVs were recovered by microinjection of recombinant DNA into NIH/3T3 cells and tested for their leukemogenic potential by inoculation into NIH/Swiss newborn mice. Parental Moloney MuLV and amphotropic 4070-A MuLV induced thymic and nonthymic leukemia, respectively, when inoculated intrathymically. With chimeric MuLVs, we found that the primary determinant of leukemogenicity of Moloney and amphotropic MuLVs lies within the 1.5-kilobase-pair ClaI-PvuI long terminal repeat (LTR)-containing fragment. The presence of additional Moloney env-pol sequences with the Moloney LTR enhanced the leukemogenic potential of a chimeric MuLV significantly, indicating that these sequences were also involved in tumor development. Since parental viruses induced different forms of leukemia, we could also map the viral sequences conferring this disease specificity. We found that the 1.5-kilobase-pair ClaI-PvuI LTR-containing fragment of Moloney MuLV was necessary and sufficient for a chimeric MuLV to induce thymic leukemia. Similarly, the same LTR-containing fragment of amphotropic MuLV was necessary and sufficient for a chimeric MuLV to induce nonthymic leukemia. Therefore, our results suggest that specific sequences within this short LTR-containing fragment determine two important viral functions: the ability to transform cells in vivo (leukemic transformation) and the selection of a specific population of cells to be transformed (disease specificity).     

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.     

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.     

Tumor progression in murine leukemia virus-induced T-cell lymphomas: monitoring clonal selections with viral and cellular probes.

Clonal selections occurring during the progression of Moloney murine leukemia virus (MuLV)-induced T-cell lymphomas in mice were examined in primary and transplanted tumors by monitoring various molecular markers: proviral integration patterns, MuLV insertions near c-myc and pim-1, and rearrangements of the immunoglobulin heavy chain and beta-chain T-cell receptor genes. The results were as follows. Moloney MuLV frequently induced oligoclonal tumors with proviral insertions near c-myc or pim-1 in the independent clones. Moloney MuLV acted as a highly efficient insertional mutagen, able to activate different (putative) oncogenes in one cell lineage. Clonal selections during tumor progression were frequently marked by the acquisition of new proviral integrations. Independent tumor cell clones exhibited a homing preference upon transplantation in syngeneic hosts and were differently affected by the route of transplantation.     

Important role of the long terminal repeat of the helper Moloney murine leukemia virus in Abelson virus-induced lymphoma.

The helper virus has been shown to play a critical role in the development of lymphoma induced by the defective Abelson murine leukemia virus (A-MuLV). Indeed, A-MuLV pseudotyped with some viruses, such as the Moloney MuLV, has been shown to be highly lymphogenic, whereas A-MuLV pseudotyped with other viruses, such as the BALB/c endogenous N-tropic MuLV, has been shown to be devoid of lymphogenic potential (N. Rosenberg and D. Baltimore, J. Exp. Med. 147:1126-1141, 1978; C. D. Scher, J. Exp. Med. 147: 1044-1053, 1978). To map the viral DNA sequences encoding the determinant of the lymphogenic potential of Moloney MuLV when complexed with A-MuLV, we constructed chimeric helper viral DNA genomes in vitro between parental cloned infectious viral DNA genomes from Moloney MuLV and from BALB/c endogenous N-tropic MuLV. Chimeric helper MuLVs, recovered after transfection of NIH 3T3 cells were used to rescue A-MuLV, and the pseudotypes were inoculated into newborn NIH Swiss, CD-1, and SWR/J mice to test their lymphogenic potential. We found that a 0.44-kilobase-pair PstI-KpnI long terminal repeat-containing fragment from the Moloney MuLV was sufficient to confer some, but not complete, lymphogenic potential to a chimeric virus (p7M2) in NIH Swiss and SWR/J mice, but not in CD-1 mice. The addition of the 3'-end env sequences (comprising the carboxy terminus of gp70 and all p15E) to the U3 long terminal repeat sequences restored the full lymphogenic potential of the Moloney MuLV. Our data indicate that the 3'-end sequences of the helper Moloney MuLV are somehow involved in the development of lymphoma induced by A-MuLV. The same sequences have previously been found to harbor the determinant of leukemogenicity and of disease specificity of Moloney MuLV when inoculated alone.     

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.     

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.     

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.     

Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat.

The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts (E. Rassart, M. Shang, Y. Boie, and P. Jolicoeur, J. Virol. 58:96-106, 1986). To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR.     

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.     

An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer.

Direct RNA-PCR analyses of T-cell lymphomas that developed in rhesus macaques during a gene transfer experiment revealed the presence of several different recombinant murine leukemia viruses (MuLV). Most prominent was the expected MuLV recombinant, designated MoLTRAmphoenv in which the amphotropic env of the helper packaging virus was joined to the long terminal repeat (LTR) of the Moloney MuLV-derived vector. This retrovirus does not exist in nature. An additional copy of the core enhancer acquired from the vector LTR may have augmented the replicative properties of MoLTRAmphoenv MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of mink cell focus-forming MuLV elements, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of an array of MuLV-related structures in a primate gene transfer recipient demands ever-vigilant scrutiny for the existence of transmissible retroviral elements and replication-competent viruses possessing altered tropic or growth properties in packaging cells producing retroviral vectors.     

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.     

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.     

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.     

Tandemization of a Subregion of the Enhancer Sequences from SRS 19-6 Murine Leukemia Virus Associated with T-Lymphoid but Not Other Leukemias

Most simple retroviruses induce tumors of a single cell type when infected into susceptible hosts. The SRS 19-6 murine leukemia virus (MuLV), which originated in mainland China, induces leukemias of multiple cellular origins. Indeed, infected mice often harbor more than one tumor type. Since the enhancers of many MuLVs are major determinants of tumor specificity, we tested the role of the SRS 19-6 MuLV enhancers in its broad disease specificity. The enhancer elements of the Moloney MuLV (M-MuLV) were replaced by the 170-bp enhancers of SRS 19-6 MuLV, yielding the recombinants DeltaMo+SRS(+) and DeltaMo+SRS(-) M-MuLV. M-MuLV normally induces T-lymphoid tumors in all infected mice. Surprisingly, when neonatal mice were inoculated with DeltaMo+SRS(+) or DeltaMo+SRS(-) M-MuLV, all tumors were of T-lymphoid origin, typical of M-MuLV rather than SRS 19-6 MuLV. Thus, the SRS 19-6 MuLV enhancers did not confer the broad disease specificity of SRS 19-6 MuLV to M-MuLV. However, all tumors contained DeltaMo+SRS M-MuLV proviruses with common enhancer alterations. These alterations consisted of tandem multimerization of a subregion of the SRS 19-6 enhancers, encompassing the conserved LVb and core sites and adjacent sequences. Moreover, when tumors induced by the parental SRS 19-6 MuLV were analyzed, most of the T-lymphoid tumors had similar enhancer alterations in the same region whereas tumors of other lineages retained the parental SRS 19-6 MuLV enhancers. These results emphasize the importance of a subregion of the SRS 19-6 MuLV enhancer in induction of T-cell lymphoma. The relevant sequences were consistent with crucial sequences for T-cell lymphomagenesis identified for other MuLVs such as M-MuLV and SL3-3 MuLV. These results also suggest that other regions of the SRS 19-6 MuLV genome contribute to its broad leukemogenic spectrum.     

Long-range mapping of Mis-2, a common provirus integration site identified in murine leukemia virus-induced thymomas and located 160 kilobase pairs downstream of Myb.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.     

Neurotropic Cas-BR-E murine leukemia virus harbors several determinants of leukemogenicity mapping in different regions of the genome.

The infectious virus derived from the molecularly cloned genome of the neurotropic ecotropic murine Cas-BR-E retrovirus was previously shown to have retained the ability to induce hind-limb paralysis and leukemia when inoculated into susceptible mice (P. Jolicoeur, N. Nicolaiew, L. DesGroseillers, and E. Rassart, J. Virol. 45:1159-1163, 1983). To map the viral sequences encoding the leukemogenic determinant(s) of this virus, we used chimeric viral genomes constructed in vitro between cloned viral DNAs from the leukemogenic Cas-BR-E murine leukemia virus (MuLV) and from the related nonleukemogenic amphotropic 4070-A MuLV. Infectious chimeric MuLVs, recovered from NIH 3T3 cells microinjected with these DNAs, were inoculated into newborn NIH Swiss, SIM.S, and SWR/J mice to test their leukemogenic potential. We found that each chimeric MuLV, harboring either the long terminal repeat, the gag-pol, or the pol-env region of the Cas-BR-E MuLV genome, was leukemogenic, indicating that this virus harbors several determinants of leukemogenicity mapping in different regions of its genome. This result suggests that the amphotropic 4070-A MuLV has multiple regions along its genome which prevent the expression of its leukemogenic phenotype, and it also shows that substitution of only one of these regions for Cas-BR-E MuLV sequences is sufficient to make it leukemogenic.     

Characterization of a progressive neurodegenerative disease induced by a temperature-sensitive Moloney murine leukemia virus infection.

A progressive neurodegenerative disease occurred following infection of mice with a temperature-sensitive (ts) isolate of Moloney (Mo) murine leukemia virus (MuLV), ts Mo BA-1 MuLV. This NB-tropic ecotropic MuLV, which was ts for a late function, induced a syndrome of tremor, weakness of the hind limbs, and spasticity following infection of several strains of laboratory neonatal mice, including NFS, C3H/He, CBA, SJL, and BALB/c. The latent period of 8 to 16 weeks was considerably longer than that observed for the acute paralytic diseases observed following neonatal infection with other ts Mo-MuLV, rat-passaged Friend MuLV, and some wild mouse ecotropic MuLVs. Spongiform pathology without inflammation and degeneration of neurons devoid of budding virions occurred in the cerebellar grey matter, brain stem, and upper spinal cord; but lower spinal cord anterior horn cells were less obviously affected than in other MuLV-associated neuroparalytic syndromes. ts Mo BA-1 MuLV differed from other ts Mo-MuLV mutants that are capable of inducing a neuroparalytic syndrome in that while infected nervous system tissue contained high levels of MuLV p30 and gp70, no evidence of precursor accumulation or abnormal processing of MuLV p30 or gp70 could be demonstrated. The localization of virus within the nervous system suggests that direct neuronal infection may not be the etiologic mechanism in this MuLV-induced neurodegenerative disease.     

Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.     

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.