Hematologic disease induced in BALB/c mice by a bcr-abl retrovirus is influenced by the infection conditions.

Irradiated mice reconstituted with bone marrow cells infected with a retrovirus carrying the bcr-abl oncogene of human chronic myeloid leukemia are subject to a range of neoplastic hematopoietic diseases, both myeloid and lymphoid. Comparison of DBA/2 and C57BL/6 mice has revealed a marked strain difference in susceptibility to the various tumor types. The present study, performed with BALB/c mice, indicates that the kinetics and nature of the induced disease can be modulated by the infection procedure, as well as the genetic background, and that retroviral regulatory sequences may influence the outcome. A distinctive clonal myeloproliferative disorder, somewhat akin to chronic myeloid leukemia but with prominent erythroid and mast cell components, as well as granulocytic excess, was characterized.     

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.     

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.     

Bone marrow depletion by 89Sr complements a preleukemic defect in a long terminal repeat variant of Moloney murine leukemia virus.

We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo + PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo + PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo + PyF101 Mo-MuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo + PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo + PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.     

Novel insights into the pathogenesis of the Graffi murine leukemia retrovirus

The Graffi murine leukemia virus (MuLV) was isolated in 1954 by Arnold Graffi, who characterized it as a myeloid leukemia-inducing retrovirus. He and his team, however, soon observed the intriguing phenomenon of hematological diversification, which corresponded to a decrease of myeloid leukemias and an increase of other types of leukemias. Recently, we derived two different molecular clones corresponding to ecotropic nondefective genomes that were named GV-1.2 and GV-1.4. The induced leukemias were classified as myeloid based on morphological analysis of blood smears. In this study, we further characterized the two variants of the Graffi murine retrovirus, GV-1.2 and GV-1.4, in three different strains of mice. We show that the Graffi MuLV is a multipotent retrovirus capable of inducing both lymphoid (T- and B-cell) and nonlymphoid (myeloid, erythroid, megakaryocytic) leukemia. Many of these are very complex with concomitant expression of different hematopoietic lineages. Interestingly, a high percentage of megakaryocytic leukemias, a type of leukemia rarely observed with MuLVs, arise in the FVB/n strain of mice. The genetic backgrounds of the different strains of mice influence greatly the results. Furthermore, the enhancer region, different for GV-1.2 and GV-1.4, plays a pivotal role in the disease specificity: GV-1.2 induces more lymphoid leukemias, and GV-1.4 induces more nonlymphoid ones.     

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 2.4-kilobase-pair fragment of the Friend murine leukemia virus genome contains the sequences responsible for friend murine leukemia virus-induced erythroleukemia.

Friend murine leukemia virus (F-MuLV) is a replication-competent, ecotropic, NB-tropic retrovirus which produces a rapidly fatal erythroleukemia in susceptible strains of mice. We previously molecularly cloned the entire F-MuLV genome. Transfection of this cloned DNA into NIH 3T3 mouse fibroblasts produces a virus with the same leukemia-inducing characteristics as F-MuLV. To identify which portion of the F-MuLV genome is responsible for causing leukemia, we made recombinant viruses between subgenomic fragments of F-MuLV DNA and another retrovirus--Amphotroph clone 4070. Amphotroph clone 4070 is a replication-competent, amphotrophic, N-tropic virus which does not produce any detectable malignancy in mice. A 2.4-kilobase-pair fragment of F-MuLV DNA was isolated. This DNA fragment encompassed approximately 700 base pairs from the 3' end of the F-MuLV pol gene and 1.7 kilobase pairs of the env gene including all of gp70 and the N-terminal four-fifths of p15E. A molecularly cloned fragment of Amphotroph DNA was ligated to the 2.4-kilobase-pair F-MuLV DNA, and an 8.3-kilobase-pair hybrid F-MuLV-Amphotroph DNA was subcloned into a new plasmid (p5a25-H). Transfection of p5a25-H DNA into fibroblasts resulted in the production of a replication-competent, ecotropic, N-tropic retrovirus--5a25-H virus. Inoculation of this virus into newborn NIH Swiss mice caused leukemia within 4 to 6 months. The disease caused by 5a25-H was pathologically and histologically indistinguishable from the disease caused by F-MuLV. We conclude that the F-MuLV sequences needed to cause disease are contained in these 2.4 kilobase pairs of DNA.     

Neurodegenerative disease induced by the wild mouse ecotropic retrovirus is markedly accelerated by long terminal repeat and gag-pol sequences from nondefective Friend murine leukemia virus.

The wild mouse ecotropic retrovirus (WM-E) induces a spongiform neurodegenerative disease in mice after a variable incubation period of 2 months to as long as 1 year. We isolated a molecular clone of WM-E (15-1) which was weakly neurovirulent (incidence, 8%) but was highly leukemogenic (incidence, 45%). Both lymphoid and granulocytic leukemias were observed, and these leukemias were often neuroinvasive. A chimeric virus was constructed containing the env and 3' pol sequences of 15-1 and long terminal repeat (LTR), gag, and 5' pol sequences from a clone of Friend murine leukemia virus (FB29). FB29 has been shown previously to replicate to high levels in the central nervous system (CNS) but is not itself neurovirulent. This finding was confirmed at the DNA level in the current study. Surprisingly, intraperitoneal inoculation of neonatal IRW mice with the chimeric virus (FrCasE) caused an accelerated neurodegenerative disease with an incubation period of only 16 days and was uniformly fatal by 23 days postinoculation. Introduction of the LTR of 15-1 into the FrCasE genome yielded a virus (FrCasEL) with a degree of neurovirulence intermediate between those of 15-1 and FrCasE. No differences were found in the levels of viremia or the relative levels of viral DNA in the spleens of mice inoculated with 15-1, FrCasE, or FrCasEL. However, the levels of viral DNA in the CNS correlated with the relative degrees of neurovirulence of the respective viruses (FrCasE greater than FrCasEL greater than 15-1). Thus, the env and 3' pol sequences of WM-E (15-1) were required for neurovirulence, but elements within the LTR and gag-pol regions of FB29 had a profound influence on the level of CNS infection and the rate of development of neurodegeneration.     

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.     

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.     

Analysis of the Disease Potential of a Recombinant Retrovirus Containing Friend Murine Leukemia Virus Sequences and a Unique Long Terminal Repeat from Feline Leukemia Virus

We have molecularly cloned a feline leukemia virus (FeLV) (clone 33) from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV proviruses from other cats with AML, contains an unusual structure in its U3 region upstream of the enhancer (URE) consisting of three tandem direct repeats of 47 bp. To test the disease potential and specificity of this unique FeLV LTR, we replaced the U3 region of the LTR of the erythroleukemia-inducing Friend murine leukemia virus (F-MuLV) with that of FeLV clone 33. When the resulting virus, F33V, was injected into newborn mice, almost all of the mice eventually developed hematopoietic malignancies, with a significant percentage being in the myeloid lineage. This is in contrast to mice injected with an F-MuLV recombinant containing the U3 region of another FeLV that lacks repetitive URE sequences, none of which developed myeloid malignancies. Examination of tumor proviruses from F33V-infected mice failed to detect any changes in FeLV U3 sequences other than that in the URE. Like F-MuLV-infected mice, those infected with the F-MuLV/FeLV recombinants were able to generate and replicate mink cell focus-inducing viruses. Our studies are consistent with the idea that the presence of repetitive sequences upstream of the enhancer in the LTR of FeLV may favor the activation of this promoter in myeloid cells and contribute to the development of malignancies in this hematopoietic lineage.     

Substitution of feline leukemia virus long terminal repeat sequences into murine leukemia virus alters the pattern of insertional activation and identifies new common insertion sites

The recombinant retrovirus, MoFe2-MuLV (MoFe2), was constructed by replacing the U3 region of Moloney murine leukemia virus (M-MuLV) with homologous sequences from the FeLV-945 LTR. NIH/Swiss mice neonatally inoculated with MoFe2 developed T-cell lymphomas of immature thymocyte surface phenotype. MoFe2 integrated infrequently (0 to 9%) near common insertion sites (CISs) previously identified for either parent virus. Using three different strategies, CISs in MoFe2-induced tumors were identified at six loci, none of which had been previously reported as CISs in tumors induced by either parent virus in wild-type animals. Two of the newly identified CISs had not previously been implicated in lymphoma in any retrovirus model. One of these, designated 3-19, encodes the p101 regulatory subunit of phosphoinositide-3-kinase-gamma. The other, designated Rw1, is predicted to encode a protein that functions in the immune response to virus infection. Thus, substitution of FeLV-945 U3 sequences into the M-MuLV long terminal repeat (LTR) did not alter the target tissue for M-MuLV transformation but significantly altered the pattern of CIS utilization in the induction of T-cell lymphoma. These observations support a growing body of evidence that the distinctive sequence and/or structure of the retroviral LTR determines its pattern of insertional activation. The findings also demonstrate the oligoclonal nature of retrovirus-induced lymphomas by demonstrating proviral insertions at CISs in subdominant populations in the tumor mass. Finally, the findings demonstrate the utility of novel recombinant retroviruses such as MoFe2 to contribute new genes potentially relevant to the induction of lymphoid malignancy.     

Therapeutic Effect of a Gag-Nuclease Fusion Protein against Retroviral Infection In Vivo

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.     

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).     

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.