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.     

Contribution of the gag and pol sequences to the leukemogenicity of Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) is a highly leukemogenic replication-competent murine retrovirus. Both the F-MuLV envelope gene and the long terminal repeat (LTR) contribute to its pathogenic phenotype (A. Oliff, K. Signorelli, and L. Collins, J. Virol. 51:788-794, 1984). To determine whether the F-MuLV gag and pol genes also possess sequences that affect leukemogenicity, we generated recombinant viruses between the F-MuLV gag and pol genes and two other murine retroviruses, amphotrophic clone 4070 (Ampho) and Friend mink cell focus-inducing virus (Fr-MCF). The F-MuLV gag and pol genes were molecularly cloned on a 5.8-kilobase-pair DNA fragment. This 5.8-kilobase-pair F-MuLV DNA was joined to the Ampho envelope gene and LTR creating a hybrid viral DNA, F/A E+L. A second hybrid viral DNA, F/Fr ENV, was made by joining the 5.8-kilobase-pair F-MuLV DNA to the Fr-MCF envelope gene plus the F-MuLV LTR. F/A E+L and F/Fr ENV DNAs generated recombinant viruses upon transfection into NIH 3T3 cells. F/A E+L virus (F-MuLV gag and pol, Ampho env and LTR) induced leukemia in 20% of NIH Swiss mice after 6 months. Ampho-infected mice did not develop leukemia. F/Fr ENV virus (F-MuLV gag and pol, Fr-MCV env, F-MuLV LTR) induced leukemia in 46% of mice after 3 months. Recombinant viruses containing the Ampho gag and pol, Fr-MCF env, and F-MuLV LTR caused leukemia in 38% of mice after 6 months. We conclude that the F-MuLV gag and pol genes contain sequences that contribute to the pathogenicity of murine retroviruses. These sequences can convert a nonpathogenic virus into a leukemia-causing virus or increase the pathogenicity of viruses that are already leukemogenic.     

Regions of the Moloney murine leukemia virus genome specifically related to induction of promonocytic tumors.

Moloney murine leukemia virus (MuLV) can be a potent inducer of promonocytic leukemias in mice that are undergoing a chronic inflammatory response. The neoplasms are, at least in part, associated with insertional mutagenesis of the c-myb locus. Evidence is presented for the existence of at least two genetic elements of the virus that are crucial to induction of this disease but are not required for viral replication in hematopoietic tissues or induction of lymphoid disease. These genetic elements were detected by testing the pathogenicity of recombinants between Moloney and Friend MuLVs, the latter of which is nonleukemic to myeloid cells under these conditions, and by testing Moloney MuLV-based viruses that have nonretroviral sequences inserted at specific endonuclease sites in their long terminal repeats (LTRs). Analysis of the Moloney/Friend recombinants showed that there are sequences within the structural gene domain of Moloney, but not Friend, MuLV that are necessary for promonocytic leukemia, whereas the LTRs of the MuLVs are equally effective for promonocytic tumor formation and insertional mutagenesis of the c-myb gene. Experiments with viruses which were mutagenized in the LTR by insertions demonstrated that there is a specific genetic element in the U3 region of the LTR of Moloney MuLV, upstream of the 75-base-pair enhancer which, when interrupted, results in loss of leukemogenicity for cells in the monocytic lineage but not cells in the lymphoid lineage. We conclude, therefore, that promonocytic leukemia induction, in Moloney MuLV-infected mice undergoing a chronic inflammatory response, requires specific sequences in the structural gene region of Moloney MuLV as well as other sequences in the regulatory region of the virus.     

A Moloney murine leukemia virus-based retrovirus with 4070A long terminal repeat sequences induces a high incidence of myeloid as well as lymphoid neoplasms

Retroviruses can be used to accelerate hematopoietic cancers predisposed to neoplastic disease by prior genetic manipulations such as in transgenic or knockout mice. The virus imparts a second neoplastic "hit," providing evidence that the initial hit is transforming. In the present study, a unique retrovirus was developed that can induce a high incidence of myeloid disease and has a broad host range. This agent is a Moloney murine leukemia virus (Mo-MuLV)-based virus that has most of the U3 region of the long terminal repeat (LTR) replaced with that of retrovirus 4070A. Like Mo-MuLV, this virus, called MOL4070LTR, is NB-tropic and not restricted by Fv1 allelles. MOL4070LTR causes myeloid leukemias in ca. 50% of mice, a finding in contrast to Mo-MuLV, which induces almost exclusively lymphoid disease. The data suggest that the LTR of the 4070A virus expands the tissue tropism of the disease to the myeloid lineage. Interesting, MCF recombinant envelope was expressed in the lymphoid but not the myeloid neoplasms of BALB/c mice. This retrovirus has the potential for accelerating myeloid disease in genetically engineered mice.     

The envelope gene and long terminal repeat sequences contribute to the pathogenic phenotype of helper-independent Friend viruses.

Friend murine leukemia virus (F-MuLV) and Friend mink cell focus-inducing virus (Fr-MCF) are helper-independent murine retroviruses which induce a rapidly fatal erytholeukemia in NIH Swiss mice. Amphotropic clone 4070 (Ampho) is a murine retrovirus which does not cause leukemia in these animals. Mice inoculated with Ampho, an Fr-MCF/Ampho pseudotype, or F-MuLV developed leukemia in 0, 50, and 100% of animals, respectively. To identify the F-MuLV and Fr-MCF sequences responsible for leukemia, we constructed hybrid viral genomes between these viruses and Ampho, using subgenomic fragments of molecularly cloned viral DNA. Transfection of these hybrid viral DNAs into fibroblasts produces recombinant retroviruses. These new viruses are assayed in vivo for their ability to cause leukemia. Recombinant viruses constructed between the Ampho genome and the Fr-MCF envelope gene do not cause leukemia. Similarly, viruses constructed by using either the Fr-MCF long terminal repeat U3 region or the F-MuLV long terminal repeat U3 region and the remainder of the Ampho genome do not cause leukemia. However, if the Fr-MCF envelope gene plus the Fr-MCF U3 region are joined to Ampho, the resulting virus causes erythroleukemia in 14% of mice. Recombinant viruses made between the Fr-MCF envelope gene, the F-MuLV U3 region, and the remainder of the Ampho genome cause erythroleukemia in 38% of mice. This study demonstrates that both the envelope gene of Fr-MCF and the U3 regions of Fr-MCF and F-MuLV contain sequences which contribute to the leukemic phenotype of helper-independent Friend viruses.     

Structure and function of the long terminal repeats of feline leukemia viruses derived from naturally occurring acute myeloid leukemias in cats.

Long terminal repeats of feline leukemia viruses cloned from feline acute myeloid leukemias frequently contained direct repeats of 40 to 74 bp in the upstream region of the enhancer (URE). The repetitive URE conferred an enhancer function upon gene expression in myeloid cells, suggesting its association with tumorigenic potential in myeloid cells.     

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.     

Common proviral integration region on mouse chromosome 7 in lymphomas and myelogenous leukemias induced by Friend murine leukemia virus.

Friend murine leukemia virus (F-MuLV) induces a variety of hematopoietic neoplasms 2 to 12 months after inoculation into newborn mice. These neoplasms are clonal or oligoclonal and contain a small number of F-MuLV insertions in high-molecular-weight DNA. To investigate whether different tumors have proviral insertions in the same region, a provirus-cellular DNA junction fragment from an F-MuLV-induced myelogenous leukemia was cloned in lambda gtWES, and a portion of the flanking cellular DNA sequence was used in blot-hybridization studies of 34 additional F-MuLV-induced neoplasms. Three of these additional neoplasms (one myelogenous leukemia and two lymphomas) were found to have altered copies of the flanking cellular sequence. Restriction enzyme analysis of genomic DNA from these tumors revealed that in each case a proviral copy of F-MuLV had inserted into the same 1.5-kilobase region; all proviruses had the same orientation. Using mouse-Chinese hamster somatic cell hybrids, we mapped this common integration region, designated Fis-1, to mouse chromosome 7. Fis-1 is distinct from three oncogenes on mouse chromosome 7, Ha-ras, fes, and Int-2, based on restriction enzyme analysis and blot hybridization. Therefore, Fis-1 appears to be a novel sequence implicated in both lymphoid and myeloid leukemias induced by F-MuLV.     

Rearrangements and insertions in the Moloney murine leukemia virus long terminal repeat alter biological properties in vivo and in vitro.

The effects of rearrangement and insertion of sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) were investigated. The alterations were made by recombinant DNA manipulations on a plasmid subclone containing an M-MuLV LTR. Promoter activity of altered LTRs was measured by fusion to the bacterial chloramphenicol acetyltransferase gene, followed by transient expression assay in NIH 3T3 cells. M-MuLV proviral organizations containing the altered LTRs were also generated, and infectious virus was recovered by transfection. Infectivity of the resulting virus was quantified by XC plaque assay, and pathogenicity was determined by inoculating neonatal NIH Swiss mice. Inversion of sequences in the U3 region containing the tandemly repeated enhancer sequences (-150 to -353 base pairs [bp]) reduced promoter activity approximately fivefold in the transient-expression assays. Infectious virus containing the inverted sequences (Mo- M-MuLV) showed a 20-fold reduction in relative infectivity compared with wild-type M-MuLV, but the virus still induced thymus-derived lymphoblastic lymphoma or leukemia in mice, with essentially the same kinetics as for wild-type M-MuLV. We previously derived an M-MuLV which carried inserted enhancer sequences from the F101 strain of polyomavirus (Mo + PyF101 M-MuLV) and showed that this virus is nonleukemogenic. In Mo + PyF101 M-MuLV, the PyF101 sequences were inserted between the M-MuLV promoter and the M-MuLV enhancers (at -150 bp). A new LTR was generated in which the PyF101 sequences were inserted to the 5' side of the M-MuLV enhancers (at -353 bp, PyF101 + Mo M-MuLV). The PyF101 + Mo LTR exhibited promoter activity similar (40 to 50%) to that of wild-type M-MuLV, and infectious PyF101 + Mo M-MuLV had high infectivity on NIH 3T3 cells (50% of wild type). In contrast to the nonleukemogenic Mo + PyF101 M-MuLV, PyF101 + Mo M-MuLV induced leukemia with kinetics similar to that of wild-type M-MuLV. Thus, the position of the PyF101 sequences relative to the M-MuLV LTR affected the biological behavior of the molecular construct. Furthermore, PyF101 + Mo M-MuLV induced a different spectrum of neoplastic disease. In comparison with wild-type M-MuLV, which induces a characteristic thymus-derived lymphoblastic lymphoma with extremely high frequency, PyF101 + Mo M-MuLV was capable of inducing both acute myeloid leukemia or thymus-derived lymphoblastic lymphoma, or both. Tumor DNA from both the PyF101 + Mo- and Mo- M-MuLV-inoculated animals contained recombinant proviruses with LTRs that differed from the initially inoculated virus.     

Sequence-Specific and/or Stereospecific Constraints of the U3 Enhancer Elements of MCF 247-W Are Important for Pathogenicity

The oncogenic potential of many nonacute retroviruses is dependent on the duplication of the enhancer sequences present in the unique 3′ (U3) region of the long terminal repeat (LTR). In a molecular clone (MCF 247-W) of the murine leukemia virus MCF 247, a leukemogenic mink cell focus-inducing (MCF) virus, the U3 enhancer sequences are tandemly repeated in the LTR. We mutated the enhancer region of MCF 247-W to test the hypothesis that the duplicated enhancer sequences of this virus have a sequence-specific and/or a stereospecific role in enhancer function required for transformation. In one virus, we inserted 14 nucleotide bp into the novel sequence generated at the junction of the two enhancers to generate an MCF virus with an interrupted enhancer region. In the second virus, only one copy of the enhancer sequences was present. This second virus also lacked the junction sequence present between the two enhancers of MCF 247-W. Both viruses were less leukemogenic and had a longer mean latency period than MCF 247-W. These data indicate that the sequence generated at the junction of the two enhancers and/or the stereospecific arrangement of the two enhancer elements are required for the full oncogenic potential of MCF 247-W. We analyzed proviral LTRs within the c-myc locus in tumor DNAs from mice injected with the MCF virus with the interrupted enhancer region. Some of the proviral LTRs integrated upstream of c-myc contain enhancer regions that are larger than those of the injected virus. These results are consistent with the suggestion that the virus with an interrupted enhancer changes in vivo to perform its role in the transformation of T cells.     

Tissue-specific replication of Friend and Moloney murine leukemia viruses in infected mice.

We have studied the replication of ecotropic murine leukemia viruses (MuLV) in the spleens and thymuses of mice infected with the lymphocytic leukemia-inducing virus Moloney MuLV (M-MuLV), with the erythroleukemia-inducing virus Friend MuLV (F-MuLV), or with in vitro-constructed recombinants between these viruses in which the long terminal repeat (LTR) sequences have been exchanged. At 1 week after infection both the parents and the LTR recombinants replicated predominantly in the spleens with only low levels of replication in the thymus. At 2 weeks after infection, the patterns of replication in the spleens and thymuses were strongly influenced by the type of LTR. Viruses containing the M-MuLV LTR exhibited a remarkable elevation in thymus titers which frequently exceeded the spleen titers, whereas viruses containing the F-MuLV LTR replicated predominantly in the spleen. In older preleukemic mice (5 to 8 weeks of age) the structural genes of M-MuLV or F-MuLV predominantly influenced the patterns of replication. Viruses containing the structural genes of M-MuLV replicated efficiently in both the spleen and thymus, whereas viruses containing the structural genes of F-MuLV replicated predominantly in the spleen. In leukemic mice infected with the recombinant containing F-MuLV structural genes and the M-MuLV LTR, high levels of virus replication were observed in splenic tumors but not in thymic tumors. This phenotypic difference suggested that tumors of the spleen and thymus may have originated by the independent transformation of different cell types. Quantification of polytropic MulVs in late-preleukemic mice infected with each of the ecotropic MuLVs indicated that the level of polytropic MuLV replication closely paralleled the level of replication of the ecotropic MuLVs in all instances. These studies indicated that determinants of tissue tropism are contained in both the LTR and structural gene sequences of F-MuLV and M-MuLV and that high levels of ecotropic or polytropic MuLV replication, per se, are not sufficient for leukemia induction. Our results further suggested that leukemia induction requires a high level of virus replication in the target organ only transiently during an early preleukemic stage of disease.     

Different abilities of Friend murine leukemia virus (MuLV) and Moloney MuLV to induce promonocytic leukemia are due to determinants in both psi-gag-PR and env regions.

Moloney murine leukemia virus (M-MuLV) is capable of inducing promonocytic leukemia in 50% of adult BALB/c mice that have received peritoneal injections of pristane, but Friend MuLV strain 57 (F-MuLV) is nonleukemogenic under similar conditions. It was shown earlier that these differences could not be mapped to the U3 region of the virus long terminal repeat, indicating the probable influence of structural genes and/or R-U5 sequences. In this study, reciprocal chimeras containing exchanged structural genes and R-U5 sequences from these two closely related viruses were analyzed for differences in ability to induce disease. Results showed that two regions of F-MuLV, psi-gag-PR and env, when substituted for those of M-MuLV were dramatically disease attenuating. The 5'-most region, which is widely distributed, overlaps with the 5' end of the env intron and includes the RNA packaging region, psi, the entire gag coding region, and the viral protease coding region (PR) of pol. It was also found that reciprocal constructs having substitutions of both of these regions of M-MuLV in an F-MuLV background allowed full reestablishment of promonocytic leukemia. These leukemias were positive for c-myb rearrangements which are characteristic of M-MuLV-induced promonocytic leukemias. Neither region alone, however, was sufficient to produce disease with a greater incidence than 13%. Further studies demonstrated that the inability of viruses with psi, gag, PR, or env sequences from F-MuLV to induce leukemia in this model system was not due to their inability to replicate in hematopoietic tissue, to integrate into the c-myb locus early on after infection in vivo, or to express gag-myb mRNA characteristic of M-MuLV-induced preleukemic cells and acute leukemia.     

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.     

Origin of pathogenic determinants of recombinant murine leukemia viruses: analysis of Bxv-1-related xenotropic viruses from CWD mice.

The acquisition of U3 region sequences derived from the endogenous xenotropic provirus Bxv-1 appears to be an important step in the generation of leukemogenic recombinant viruses in AKR, HRS, C58, and some CWD mice. We report here that each of three CWD lymphomas produced infectious xenotropic murine leukemia virus related to Bxv-1. In Southern blot experiments, these proviruses hybridized to probes that were specific for the xenotropic envelope and Bxv-1 U3 region sequences. Nucleotide sequence analysis of a cloned CWD xenotropic provirus, CWM-S-5X, revealed that the envelope gene was closely related to but distinct from those of other known xenotropic viruses. In addition, the U3 region of CWM-S-5X contained a viral enhancer sequence that was identical to that found in MCF 247, a recombinant AKR virus that is thought to contain the Bxv-1 enhancer. Finally, restriction enzyme sites in the CWM-S-5X provirus were analogous to those reported within Bxv-1. These results establish that the virus progeny of Bxv-1 have the potential to donate pathogenic enhancer sequences to recombinant polytropic murine leukemia viruses. Interestingly, the three CWD polytropic viruses that were isolated from the same tumor cells that produced the Bxv-1-like viruses had not incorporated Bxv-1 sequences into the U3 region.     

Escape from in vivo restriction of Moloney mink cell focus-inducing viruses driven by the Mo+PyF101 long terminal repeat (LTR) by LTR alterations.

Mo+PyF101 M-MuLV is a variant Moloney murine leukemia virus containing polyomavirus F101 enhancers inserted just downstream from the M-MuLV enhancers in the long terminal repeat (LTR). The protein coding sequences for this virus are identical to those of M-MuLV. Mo+PyF101 M-MuLV induces T-cell disease with a much lower incidence and longer latency than wild-type M-MuLV. We have previously shown that Mo+PyF101 M-MuLV is defective in preleukemic events induced by wild-type M-MuLV, including splenic hematopoietic hyperplasia, bone marrow depletion, and generation of recombinant mink cell focus-inducing viruses (MCFs). We also showed that an M-MCF virus driven by the Mo+PyF101 LTR is infectious in vitro but does not propagate in mice. However, in these experiments, when a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF was inoculated into newborn NIH Swiss mice, they died of T-cell leukemia at times almost equivalent to those induced by wild-type M-MuLV. Tumor DNAs from Mo+PyF101 M-MuLV-Mo+PyF101 MCF-inoculated mice were examined by Southern blot analysis. The predominant forms of Mo+PyF101 MCF proviruses in these tumors contained added sequences in the U3 region of the LTR. The U3 regions of representative tumor-derived variant Mo+PyF101 MCFs were cloned by polymerase chain reaction amplification, and sequencing indicated that they had acquired an additional copy of the M-MuLV 75-bp tandem repeat in the enhancer region. NIH 3T3 cell lines infected with altered viruses were obtained from representative Mo+PyF101 M-MuLV-Mo+PyF101 MCF-induced tumors, and mice were inoculated with the recovered viruses. Leukemogenicity was approximately equivalent to that in the original Mo+PyF101 M-MuLV-Mo+PyF101 MCF viral stock. Southern blot analysis on the resulting tumors now predominantly revealed loss of the polyomavirus sequences. These results suggest that the suppressive effects of the PyF101 sequences on M-MuLV-induced disease and potentially on MCF propagation were overcome in two ways: by triplication of the M-MuLV direct repeats and by loss of the polyomavirus sequences.