Genetic analysis of myeloproliferative leukemia virus, a novel acute leukemogenic replication-defective retrovirus.

The myeloproliferative leukemia virus (MPLV) is a new acute leukemogenic, nonsarcomatogenic retroviral complex that is generated during the in vivo passage of a molecularly cloned Friend ecotropic helper virus. Examination of viral RNA expression in MPLV-producing cells revealed the presence of two distinct molecular species that hybridized with a long terminal repeat or an ecotropic env-specific probe but not with a xenotropic mink cell focus-forming virus env-specific probe derived from a spleen focus-forming virus: an 8.2-kilobase species corresponding to a full-length Friend murine leukemia virus (F-MuLV) and a deleted species with a genomic size of 7.4 kilobases. This deleted virus was biologically cloned by limiting dilutions and single cell cloning in Mus dunni fibroblasts. Three nonproducer clones with normal morphologies and containing one single integrated copy of the deleted virus were superinfected with F-MuLV, Moloney murine leukemia virus, Gross murine leukemia virus, mink cell focus-forming virus (HIX), or the amphotropic 1504 murine leukemia virus. All pseudotypes caused macroscopic and microscopic abnormalities in mice that were similar to those seen in the parental stock. A comparison of the physical maps of F-MuLV and MPLV, which was deduced from the restriction enzyme digests of unintegrated proviral DNAs, indicated that the MPLV-defective genome (i) is probably derived from F-MuLV, (ii) has conserved the F-MuLV gag and pol regions, and (iii) is deleted and rearranged in the env region in a manner that is clearly distinct from that of Friend or Rauscher spleen focus-forming viruses.     

Efficient correction of mismatched bases in plasmid heteroduplexes injected into cultured mammalian cell nuclei.

Heteroduplexes were prepared from two plasmids, pRH4-14/TK and pRH5-8/TK, containing different amber mutations in the neomycin resistance gene (Neor). The Neor gene was engineered to be expressed in both bacterial and mammalian cells. A functional Neor gene conferred kanamycin resistance to bacteria and resistance to the drug G418 to mammalian cells. In addition, the plasmids contained restriction site polymorphisms which did not confer a selectable phenotype but were used to follow the pattern of correction of mismatched bases in the heteroduplexes. In a direct comparison of the efficiency of transforming mouse LMtk- cells to G418r, the injection of heteroduplexes of pRH4-14/TK-pRH5-8/TK was 10-fold more efficient than the coinjection of pRH4-14/TK and pRH5-8/TK linear plasmid DNA. In fact, injection of 5 to 10 molecules of heteroduplex DNA per cell was as efficient in transforming LMtk- cells to G418r as the injection of 5 to 10 molecules of linear plasmid DNA per cell containing a wild-type Neor gene. To determine the pattern of mismatch repair of the injected heteroduplexes, plasmids were "rescued" from the G418r cell lines. From this analysis we conclude that the generation of wild-type Neor genes from heteroduplex DNA proceeds directly by correction of the mismatched bases, rather than by alternative mechanisms such as recombination between the injected heteroduplexes. Our finding that a cell can efficiently correct mismatched bases when confronted with preformed heteroduplexes suggests that this experimental protocol could be used to study a wide range of DNA repair mechanisms in cultured mammalian cells.     

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter.

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.     

Analysis of mutant Moloney murine leukemia viruses containing linker insertion mutations in the 3'' region of pol.

Twelve linker insertion mutations have been constructed in the 3' part of the pol gene of Moloney murine leukemia virus. This region of the Moloney murine leukemia virus genome encodes IN or p46pol, which is required for integration of the retroviral DNA into the host cell chromosome. Viral proteins synthesized by these mutants were used to pseudotype a neo-containing retroviral vector. Ten of twelve linker insertion mutant pseudotypes were unable to generate stable proviruses in infected mouse cells, as measured by the formation of G418-resistant colonies. Two mutants mapping at the 3' terminus of the IN-encoding region were competent for the formation of stable vector proviruses (hundreds of G418-resistant colonies per mutant pseudotype-infected plate). Representative linker insertion mutants were also tested for the ability to synthesize viral unintegrated DNA in newly infected cells. All assayed mutants were capable of synthesizing all normal forms of viral unintegrated DNA. The structure of integrated vector proviruses generated by defective and nondefective linker insertion mutants was also analyzed. All replication-competent mutants generated normal proviruses, while the few obtainable proviruses generated by replication-defective mutants were sometimes aberrant in structure. These results argue strongly (and confirm previous data) that the IN-encoding region of pol does not play a significant role in DNA synthesis, but is absolutely required for the formation of normal proviral DNA.     

Embryonal carcinoma cells (and their somatic cell hybrids) are resistant to infection by the murine parvovirus MVM, which does infect other teratocarcinoma-derived cell lines

Minute virus of mice (MVM), a non-defective parvovirus, has been shown to infect cultures of non-pluripotent differentiated teratocarcinoma-derived cells, but pluripotent (and "nullipotent") embryonal carcinoma cells derived from the same teratocarcinoma resist MVN infection. Somatic cell hybrids between an embryonal carcinoma line and Friend erythroblastic leukemia cells are also resistant to MVM, even though Friend cells are susceptible. Among three blastocyst-derived lines tested, only one, a parietal yolk sac cell line, resists MVM infection. These results suggest that teratocarcinoma cultures may provide useful systems in which to study the cellular factors which mediate susceptibility to this teratogenic and oncolytic virus.     

fim-1 and fim-2: two new integration regions of Friend murine leukemia virus in myeloblastic leukemias.

The Friend helper murine leukemia virus (F-MuLV) induces in mice a high percentage of myeloblastic leukemias. Myeloblastic transformation is also observed after in vitro infection of long-term bone marrow cultures. To investigate the molecular events leading to the generation of myeloblastic leukemias, we first screened a panel of leukemic cells for rearrangement or amplification of known oncogenes or previously described specific integration sites. No modification of these genes was observed. Therefore, we searched for common integration sites by constructing a genomic library from a myeloblastic cell line harboring only five integrated proviruses. This library was screened with a virus-specific probe, and virus-host cellular junction fragments were subcloned. Two flanking cellular sequences corresponding to two different integrated proviruses were used to analyze additional myeloblastic leukemias. The first probe detected rearrangements in 2 of 42 myeloblastic leukemias, and the second probe detected rearrangements in 6 of 42. We demonstrated that, in each case, the rearrangement was the result of F-MuLV integration, with all proviruses in the same orientation and clustering in a region less than 3 kilobases long. The two regions, named fim-1 and fim-2, were different from 15 oncogenes tested. Rearrangements of these two regions were found in F-MuLV-induced myeloblastic leukemias but not in 20 lymphoid or erythroid leukemias induced by the same virus.     

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.     

Deletions in a recombinant retrovirus genome associated with its expression in embryonal carcinoma cells.

We analyzed embryonal carcinoma cell lines infected with a recombinant Moloney murine leukemia virus. Lines that carried but did not express the neo gene retained a provirus of LTR-gag-pol-neo-LTR, where LTR is a long terminal repeat, whereas all G418-resistant lines deleted regions that included the primer binding site and the splicing donor site. This suggested the presence of multiple inhibitory elements.     

Induction of protective immunity to Friend murine leukemia virus in genetic nonresponders to virus envelope protein

(B10.A x A/WySn)F1, H-2a/a, mice are genetic nonresponders to the envelope protein of Friend murine leukemia helper virus (F-MuLV) when immunized with a recombinant vaccinia virus expressing F-MuLV env gene. In contrast these mice can be protectively immunized against leukemogenic Friend virus complex using formalin-fixed F-MuLV virions in CFA. To determine which viral proteins were responsible for this immune protection, virion proteins prepared by SDS-PAGE and electroelution were used to immunize mice. Purified gp70 envelope protein in CFA was capable of inducing strong immune protection against the challenge with Friend virus complex in H-2a/a mice. Immunologic studies demonstrated that immunized mice developed a virus-specific T cell proliferative response and showed IgM to IgG Ig class switching of virus-neutralizing antibodies. These results indicated that genetically controlled immune nonresponsiveness to F-MuLV envelope Ag in H-2a/a mice could be overcome using denatured viral envelope protein together with a strong adjuvant.     

Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines

Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A)⁺ mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700, 13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells.Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A)⁺ RNA sequences are common to embryonal carcinoma cell polysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A)⁺ RNA fails to hybridize 7–10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.     

In vivo interactions of ecotropic and polytropic murine leukemia viruses in mixed retrovirus infections

Mixed retrovirus infections are the rule rather than the exception in mice and other species, including humans. Interactions of retroviruses in mixed infections and their effects on disease induction are poorly understood. Upon infection of mice, ecotropic retroviruses recombine with endogenous proviruses to generate polytropic viruses that utilize different cellular receptors. Interactions among the retroviruses of this mixed infection facilitate disease induction. Using mice infected with defined mixtures of the ecotropic Friend murine leukemia virus (F-MuLV) and different polytropic viruses, we demonstrate several dramatic effects of mixed infections. Remarkably, inoculation of F-MuLV with polytropic MuLVs completely suppressed the generation of new recombinant viruses and dramatically altered disease induction. Co-inoculation of F-MuLV with one polytropic virus significantly lengthened survival times, while inoculation with another polytropic MuLV induced a rapid and severe neurological disease. In both instances, the level of the polytropic MuLV was increased 100- to 1,000-fold, whereas the ecotropic MuLV level remained unchanged. Surprisingly, nearly all of the polytropic MuLV genomes were packaged within F-MuLV virions (pseudotyped) very soon after infection. At this time, only a fractional percentage of cells in the mouse were infected by either virus, indicating that the co-inoculated viruses had infected the same small subpopulation of susceptible cells. The profound amplification of polytropic MuLVs in coinfected mice may be facilitated by pseudotyping or, alternatively, by transactivation of the polytropic virus in the coinfected cells. This study illustrates the complexity of the interactions between components of mixed retrovirus infections and the dramatic effects of these interactions on disease processes.     

Friend strain of spleen focus-forming virus: a recombinant between mouse type C ecotropic viral sequences and sequences related to xenotropic virus.

The genome of the Friend strain of the spleen focus-forming virus (SFFV) has been analyzed by molecular hybridization. SFFV is composed of genetic sequences homologous to Friend type C helper virus (F-MuLV) and SFFV-specific sequences not present in F-MuLV. These SFFV-specific sequences are present in both the Friend and Rauscher strains of murine erythroleukemia virus. The SFFV-specific sequences are partially homologous to three separate strains of mouse xenotropic virus but not to several cloned mouse ecotropic viruses. Thus, the Friend strain of SFFV appears to be a recombinant between a portion of the F-MuLV genome and RNA sequences that are highly related to murine xenotropic viruses. The implications of the acquisition of the xenotropic virus-related sequences are discussed in relation to the leukemogenicity of SFFV, and a model for the pathogenicity of other murine leukemia-inducing viruses is proposed.     

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.     

Identification of a gag-encoded cytotoxic T-lymphocyte epitope from FBL-3 leukemia shared by Friend, Moloney, and Rauscher murine leukemia virus-induced tumors.

FBL-3 is a highly immunogenic murine leukemia of C57BL/6 origin induced by Friend murine leukemia virus (MuLV). Immunization of C57BL/6 mice with FBL-3 readily elicits CD8+ cytotoxic T lymphocytes (CTL) capable of lysing FBL-3 as well as syngeneic leukemias induced by Moloney and Rauscher MuLV. The aim of this current study was to identify the immunogenic epitope(s) recognized by the FBL-3-specific CD8+ CTL. A series of FBL-3-specific CD8+ CTL clones were generated from C57BL/6 mice immunized to FBL-3. The majority of CTL clones (32 of 38) were specific for F-MuLV gag-encoded antigen. By using a series of recombinant vaccinia viruses expressing full-length and truncated F-MuLV gag genes, the antigenic epitope recognized by the FBL-3 gag-specific CTL clones, as well as by bulk-cultured CTL from spleens of mice immune to FBL-3, was localized to the leader sequence of gPr80gag protein. The precise amino acid sequence of the CTL epitope in the leader sequence was identified as CCLCLTVFL (positions 85-93) by examining lysis of targets incubated with a series of synthetic leader sequence peptides. No evidence of other CTL epitopes in the gPr80gag or Pr65gag core virion structural polyproteins was found. The identity of CCLCLTVFL as the target peptide was validated by showing that immunization with the peptide elicited CTL that lysed FBL-3. The CTL elicited by the Gag peptide also specifically lysed syngeneic leukemia cells induced by Moloney and Rauscher MuLV (MBL-2 and RBL-5). The transmembrane peptide was shown to be the major gag-encoded antigenic epitope recognized by bulk-cultured CTL derived from C57BL/6 mice immunized to MBL-2 or RBL-5. Thus, the CTL epitope of FBL-3 is localized to the transmembrane anchor domain of the nonstructural Gag polyprotein and is shared by leukemia/lymphoma cell lines induced by Friend, Moloney, and Rauscher MuLV.     

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.     

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.     

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.