Different genes control the susceptibility of mice to Moloney or Abelson murine leukemia viruses.

The susceptibility of mice to lymphoma induction by Moloney or Abelson murine leukemia virus has been compared in BALB/c, C57BL/6, and BALB/cXC57BL/6 recombinant inbred strains. BALB/c mice were found to be susceptible to lymphoma induction by either virus, and C57BL/6 mice were found to be relatively resistant to lymphoma induction by either virus. The genes that control these patterns of susceptibility to each virus are not the same because susceptibility to each virus segregated independently in CXB recombinant inbred strains. We also found, as reported by Cook (W. Cook, Proc. Natl. Acad. Sci. U.S.A. 79:2917-2921, 1982), when injected intrathymically that Abelson murine leukemia virus rapidly induced thymomas in weanling B6 mice. Examination of the cellular phenotypes of the tumors induced by Abelson murine leukemia virus or by Moloney murine leukemia virus indicated that different lymphocyte subpopulations were the targets for tumor induction by each virus.     

Sequences in the U5-gag-pol region influence early and late pathogenic effects of Friend and Moloney murine leukemia viruses.

Friend replication-competent murine leukemia virus (F-MuLV), clone 57, induces a severe early hemolytic anemia and a later erythroleukemia after inoculation of newborn IRW or ICFW mice, whereas Moloney MuLV (M-MuLV) induces only lymphoid leukemia. We have shown previously that the attenuated hemolytic and erythroleukemogenic abilities of an F-MuLV variant, clone B3, were due mostly to changes in the env gene and long terminal repeat, respectively. For the present study, we derived two constructs exchanging env fragments of F-MuLV 57 and M-MuLV and compared them with two constructs described by Chatis et al. (J. Virol. 52:248-254, 1984) exchanging the U3 region of the long terminal repeat of the same parental viruses. When comparing the hemolytic effect of these constructs with those of the parent, we found that the U5-gag-pol region of F-MuLV was required for development of severe early hemolytic anemia and that, unlike the env of F-MuLV B3, the env of M-MuLV was fully competent in inducing severe early hemolytic anemia when associated with the F-MuLV U5-gag-pol and U3 regions. As expected, induction of erythroleukemia depended on the presence of the F-MuLV U3 region; however, the presence of both the U3 and U5-gag-pol regions of F-MuLV appeared to be synergistic and was associated with a more rapid appearance of erythroleukemia.     

Isolation of paralysis-inducing murine leukemia viruses from Friend virus passaged in rats.

Four clones of murine leukemia viruses (PVC-111, PVC-211, PVC-321, and PVC-441) were isolated from a paralyzed Fischer rat which had been infected with rat-passaged Friend leukemia virus. PVC-211 and PVC-321 viruses induced hind leg paralysis in rats and killed them within 1 month, and PVC-441 did so within 2 months after infection, whereas PVC-111 did not within 4 months. PVC-321 and PVC-441 but not PVC-111 virus grew well in brain and spinal cord media. The viral antigens were found often in glia cells and rarely in neurons of the rats infected with each of these PVC viruses. All of the PVC viruses induced neuronal degeneration but neither inflammation nor leukemic infiltration in the spinal cord. The isolated viruses were all ecotropic and NB-tropic. Age dependency of the susceptibility of rats to paralysis induction was observed.     

Sequences responsible for the distinctive hemolytic potentials of Friend and Moloney murine leukemia viruses are dispersed but confined to the psi-gag-PR region.

Friend and Moloney murine leukemia viruses (F- and M-MuLV) induce distinct diseases in hematopoietic tissues following inoculation of newborn mice of susceptible strains. F-MuLV induces erythroleukemia preceded by severe early hemolytic anemia; M-MuLV induces thymomas and only very mild hemolysis. The major viral determinant of severe early hemolytic anemia residues in the env gene, but sequences located outside this gene can modulate this effect. By means of genetic chimeras of F- and M-MuLV, we have found that although they are confined to the 5' portion of the env gene intron, sequences that determine the distinctive hemolytic potentials of F- and M-MuLV are widely distributed over a region spanning the RNA encapsidation domain, the gag gene, and the portion of the pol gene encoding the viral protease. Within this large region, two fragments of M-MuLV, a 1.3-kb region encoding the matrix, pp12, and capsid proteins and a 0.8-kb region encoding the nucleocapsid and the viral protease, were capable, individually, of partially attenuating the capacity of F-MuLV for induction of severe early hemolytic anemia. In association, these two fragments conferred complete attenuation. Moreover, a second pair of adjacent fragments within this large region appeared to behave cooperatively to confer complete attenuation; a 0.36-kb region roughly corresponding to the encapsidation domain, although not detectably altering hemolytic potential on its own, deepened the attenuation conferred by the adjacent 1.3-kb region. Whether capable of inducing severe early hemolytic anemia or not and despite different efficiencies of induction of recombinant polytropic viruses, all chimeric viruses retained the erythroleukemogenicity of the F-MuLV parent.     

Regulation of Moloney murine leukemia virus replication in chronically infected cells arrested at the G0/G1 phase.

The replication of Moloney murine leukemia virus (MMuLV) in chronically infected mouse cells arrested at the G0/G1 phase of the cell cycle by different procedures was investigated. MMuLV production was inhibited in glutamine- and isoleucine (Gln-Ile)-deprived G0/G1 cells. In contrast, butyric acid treatment, which efficiently arrested the cells at the G0/G1 phase of the cell cycle, did not inhibit MMuLV production. Furthermore, the inhibition of MMuLV production caused by either Gln-Ile deprivation or by interferon (IFN) treatment was overcome by butyric acid treatment. Thus, the replication of MMuLV could be dissociated from cell proliferation. The inhibition of MMuLV production in Gln-Ile-deprived cell cultures was compared to the inhibitory effect of IFN, which is known to affect budding and release of the virus. Rates of MMuLV protein synthesis were not affected in both the IFN-treated and Gln-Ile-deprived cells. However, processing of the viral polyprotein Pre65gag into p30 was blocked in the Gln-Ile-deprived cells. Furthermore, whereas in IFN-treated cells, MMuLV accumulated on the cell surface and could be released upon treatment with trypsin, in Gln-Ile-deprived cells, no virions were released by such treatment. These results indicate that in cells arrested by Gln-Ile deprivation, MMuLV is inhibited at a posttranslation step. This step appears to precede the anti-MMuLV block induced by IFN.     

Interference established in mice by infection with Friend murine leukemia virus.

Retroviral interference is manifested in chronically infected cells as a decrease in susceptibility to superinfection by virions using the same cellular receptor. The pattern of interference reflects the cellular receptor specificity of the chronically infecting retrovirus and is mediated by the viral envelope glycoprotein, which is postulated to bind competitively all cellular receptors available for viral attachment. We established retroviral interference in mice by infecting them with Friend murine leukemia virus and them measured susceptibility to superinfection by challenging the mice with the erythroproliferative spleen focus-forming virus. Infection of approximately 10% of nucleated splenocytes rendered mice 1% as susceptible to superinfection as untreated controls. The magnitude of this effect was the same in mice incapable of producing neutralizing antibodies or genetically deficient for T cells. The results indicated that retroviral interference in vivo was established rapidly with infection of a fraction of the host cell population and that the decrease in susceptibility to superinfection occurred without a detectable contribution by immunologic factors.     

Subcellular distribution of newly synthesized virus-specific polypeptides in Moloney murine leukemia virus infected cells.

Immune precipitation analysis of pulse-labeled proteins present in subcellular fractions of mouse embryo cells infected with Moloney murine leukemia virus showed the presence of anti-gp70 serum-precipitable viral envelope gene products mainly in the microsomal fractions of these cells. In contrast, anti-p30 serum-specific gag (group specific antigen) gene products were found to be distributed in similar amounts in both the microsomal and postmicrosomal supernatant fractions of pulse-labeled cells.     

Moloney murine leukemia virus tolerance in anti-CD4 monoclonal antibody-treated adult mice.

Adult mice injected with Moloney murine leukemia virus (M-MuLV) 1 day after a short term treatment with anti-CD4 mAb developed T cell lymphomas, or sarcomas when rechallenged with Moloney murine sarcoma virus (M-MSV). Neoplastic development was correlated with virus spread, as thymic and peripheral T and B lymphocytes promptly expressed M-MuLV-induced Ag after virus introduction; no virus-specific CTL generation was detected in MLTC. This failure, which was selective for M-MuLV-induced Ag, persisted throughout the life span of the mice, and was not sustained by suppressor cell activity. The frequencies of splenic virus-specific CTL precursor varied in relation to time after virus injection; in the first postinjection month, frequencies were similar to those in nonimmune control mice, while at 4 mo post-virus exposure, they showed a striking reduction. These findings indicate that a transient functional impairment of CD4+ cells at the time of retrovirus injection provided the appropriate milieu for tolerance induction in both the peripheral mature and intrathymic T cell compartments.     

Virus-specific RNA synthesis in interferon-treated mouse cells productively infected with Moloney murine leukemia virus.

Mouse cells productively infected with Moloney murine leukemia virus were treated with interferon, and intracellular virus-specific RNA was studied by hybridization with complementary DNA. The steady-state concentration of virus-specific RNA in interferon-treated cells was somewhat greater than that in untreated cells, and the rates of virus-specific RNA synthesis were approximately equal in treated and untreated cells.     

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.     

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).     

High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector.

A protocol was designed to measure the forward mutation rate over an entire gene replicated as part of a Moloney murine leukemia virus-based vector. For these studies, the herpes simplex virus thymidine kinase (tk) gene under the control of the spleen necrosis virus U3 promoter was used as target sequence since it allows selection for either the functional or the inactivated gene. Our results indicate that after one round of retroviral replication, the tk gene is inactivated at an average rate of 0.08 per cycle of replication. Southern blotting revealed that the majority of the mutant proviruses resulted from gross rearrangements and that deletions of spleen necrosis virus and tk sequences were the most frequent cause of the gene inactivation. Sequence analysis of the mutant proviruses suggested that homologous as well as nonhomologous recombination was involved in the observed rearrangements. Some mutations consisted of simple deletions, and others consisted of deletions combined with insertions. The frequency at which these mutations occurred during one cycle of retroviral replication provides evidence indicating that Moloney murine leukemia virus-based vectors may undergo genetic rearrangement at high rates. The high rate of rearrangement and its relevance for retrovirus-mediated gene transfer are discussed.     

Increased hematopoiesis in mice soon after infection by Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV), an erythroleukemogenic replication-competent retrovirus, induces leukemia in its host after a long latency. However, the early effects of infection may determine the pathway that eventually leads to malignant transformation. To determine how F-MuLV affects host cell proliferation soon after infection, BALB/c mice were inoculated with virus and then were assayed for susceptibility to appropriately pseudotyped spleen focus-forming virus (SFFV) as an indicator of erythropoietic activity. Twelve-week-old mice exposed to F-MuLV for 9 days were more susceptible (by a factor of 30) to superinfection by SFFV than were nonviremic mice. To test whether increased susceptibility was the result of increased hematopoietic activity, hematopoietic progenitors from the spleens of F-MuLV-infected mice were enumerated with a clonal culture assay. Nine days after inoculation with F-MuLV, the numbers of colony-forming progenitors increased by a factor of 4. Morphological analysis of the cultured colonies showed that erythroid, granulocytic, monocytic, and mixed granulocytic-monocytic progenitors all had increased. Thus, F-MuLV more rapidly induced a generalized increase in hematopoiesis than has previously been reported. The splenic hyperplasia induced by F-MuLV soon after infection may explain its ability to accelerate leukemogenesis in mice also infected by the polytropic Friend mink cell focus-forming virus.     

Selection of optimal polypurine tract region sequences during Moloney murine leukemia virus replication

Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.     

Selective failure of accessory cell function in Moloney murine leukemia virus-tolerant mice

Accessory cell activity of spleen cells from M-MuLV neonatally injected (tolerant) mice was studied to evaluate their ability to take part in in vitro CTL generation against tumor-associated antigens induced by M-MSV. In contrast with accessory cell activity in normal spleen cells, spleen cells from M-MuLV-tolerant mice are unable to reconstitute the in vitro virus-specific CTL generation of M-MSV immune, Ia-depleted spleen cells. A selective defect seems to characterize M-MuLV-tolerant mice as their spleens constitute a good source of accessory cells for alloantigen CTL generation.