Abelson murine leukemia virus-transformed cells that lack p53 protein synthesis express aberrant p53 mRNA species.

Cells of the Abelson murine leukemia virus-transformed line L12 that lack the p53 protein also lack polyadenylated mRNA capable of directing the synthesis of p53 in a cell-free system. Direct analysis of stable polyadenylated mRNA from a variety of cell lines shows that all p53 producers shared a common mRNA species (2.0 kilobases) which hybridized with a p53-specific cDNA probe. This species, which appears to be the mature, normal-sized p53 mRNA, was totally undetectable in L12 cells, which did not produce p53 in vivo. However, L12 cells contained two major p53-specific mRNA species of a substantially larger size (3.5 and 6.5 kilobases) than the p53-specific mRNA in the p53-producing cells. Genomic DNA analysis uncovered an apparent alteration in the 5' proximal part of only one p53 gene, which is unique to the L12 cell line. It is thus possible that the nonproducer phenotype of L12 cells is due at least in part to an alteration within a p53-specific DNA sequence. These findings define a system in which production of p53 appears to be efficiently regulated at the level of stable mRNA and which can be used to study the mechanisms controlling p53 expression in Abelson murine leukemia virus-transformed cells.     

Structure of the Abelson murine leukemia virus genome.

Virions produced from cells transformed by A-MuLV contain a 30S, 5.6 kb RNA that can be translated in a cell-free system to form the characteristic A-MuLV protein. This RNA was mapped by heteroduplex methods using DNA probes from M-MuLV, the presumed parent of A-MuLV. The overall organization of the RNA was determined by using full-length M-MuLV reverse transcribed DNA and visualizing the heteroduplexes in the electron microscope. This showed that A-MuLV and M-MuLV have homologous sequences at both ends of their RNAs but that the central portion of the A-MuLV genome is not homologous to sequences in M-MuLV RNA. A precise measure of the lengths of the shared regions was obtained by using S1 nuclease to digest hybrids between 32P-labeled M-MuLV DNA and A-MuLV RNA; the resulting fragments were analyzed for their length by electrophoresis. The regions of homology were shown to be 1320 nucleotides long at the 5' end and 730 nucleotides long at the 3' end. Thus approximately 6200 nucleotides of the approximately 8300 in M-MuLV RNA were deleted when the A-MuLV genome was formed, but an insert of 3600 nucleotides, presumably derived from the normal murine genome, was inserted in place of the deleted region.     

Thymocyte subsets transformed by Abelson murine leukemia virus.

The infectious complex of Abelson murine leukemia virus was altered by replacing its usual helper virus, Moloney leukemia virus, with radiation leukemia virus (RadLV). After intrathymic injection of the Abelson-RadLV complex, thymomas arose rapidly, as described previously for injection of the Abelson-Moloney complex. Cell lines were derived from thymomas induced by each Abelson virus complex and were classified according to normal thymus cell phenotypes. Each virus complex induced some cell lines which were like a 0.7% subpopulation of murine thymocytes in that they failed to express the Thy-1 cell-surface antigen. These lines are thus far indistinguishable from some Abelson-derived bone marrow transformants classified as pre-B cells. However, the Abelson-Moloney complex induced some cell lines which expressed low levels of Thy-1 and which shared most markers with immature blast cells of the thymic medulla, whereas the Abelson-RadLV complex induced some lines which were clearly like thymic cortex blast cells. Thus, Abelson virus can induce thymoma cell lines of at least two, and possibly three, distinct phenotypes corresponding to normal thymocyte blast subsets, the determination of which can be influenced by helper virus sequences.     

Transformation of T-lymphoid cells by Abelson murine leukemia virus.

Abelson leukemia virus (A-MuLV) is an oncogenic murine retrovirus whose genome contains sequences homologous to those of a normal cellular gene, c-abl. It has been demonstrated to cause rapid transformation of several cell types, including pre-B lymphocytes, macrophages, and fibroblasts. More recently, A-MuLV has been reported to induce thymic tumors in a mouse strain (C57BL/Ka) previously thought to be resistant to disease induction. We showed that the masses occurring after intrathymic injection of the virus were composed of lymphocytes of a previously described immature T-cell phenotype. This phenotype has been defined here by flow cytometry of 10 primary tumor samples stained with antibodies to several thymocyte differentiation antigens. Hybridization of DNAs from these tumors with v-abl, immunoglobulin mu, and T-cell antigen receptor beta-chain probes confirmed the T-lymphoid, polyclonal nature of the primary tumor cells. The primary tumors were malignant, as clearly shown by reinjection into Thy-congenic host animals. Further, four Thy- in vitro cell lines derived from three tumors differed from the majority of primary tumor cells and were similar to previously described A-MuLV-transformed pre-B cells. The consistent T-lymphoid phenotype exhibited by primary A-MuLV thymomas may represent one stage of normal thymocyte differentiation.     

Phosphorylation of the Abelson murine leukemia virus transforming protein.

The Abelson murine leukemia virus transforming gene product is a phosphorylated protein encoded by both viral and cellular sequences. This gene product has an amino-terminal region derived from the gag gene of its parent virus and a carboxyl-terminal region of (abl) derived from a normal murine cellular gene. Using a combination of partial proteolytic cleavage techniques and antisera specific for gag and abl sequences, we mapped in vivo phosphorylation sites to different regions of the protein. Phosphoproteins encoded by strain variants and transformation-defective mutants of Abelson murine leukemia virus with defined deletions in the primary sequence of the abl region were compared by two dimensional limit digest peptide mapping. Specific phosphorylation pattern differences for wild-type and mutant proteins probably represented deletions of specific phosphate acceptor sites in the abl region. An in vitro autophosphorylation activity copurified with the Abelson murine leukemia virus protein from transformation-competent strains. A peptide analysis of such in vitro reactions demonstrated that these phosphorylation sites were restricted to the amino-terminal region, and the specific sites appeared to be unrelated to the sites found on proteins phosphorylated in vivo. Thus, the autophosphorylation reaction probably correlates with an activity important in transformation, but the specific end product in vitro bears little resemblance to its function in vivo.     

Abelson murine leukemia virus variants with increased oncogenic potential.

A number of strains of Abelson murine leukemia virus (A-MuLV) with various abilities to transform cells have been identified. Among these is the A-MuLV-P90 strain, a mutant derived from A-MuLV-P120 that encodes an A-MuLV protein missing sequences that are normally present at the extreme carboxy terminus of P120 (N. Rosenberg and O. N. Witte, J. Virol. 33:340-348, 1980). This virus transforms NIH 3T3 cells efficiently but does not transform a high frequency of lymphoid cells in vitro or in vivo. In this communication, we show that of the relatively few tumors induced by A-MuLV-P90 nearly all contained new variant viruses that stably expressed either larger or smaller A-MuLV proteins. Strains that expressed larger A-MuLV proteins behaved like A-MuLV-P120 in transformation assays, whereas those expressing smaller A-MuLV proteins induced a high frequency of tumors after a short latent period in vivo but failed to transform large numbers of lymphoid cells in vitro. Thus, these latter viruses separated the requirements for in vitro transformation of lymphoid cells from those for tumor induction. All of the variants differed from A-MuLV-P90 in the carboxy-terminal region of the A-MuLV protein, suggesting that sequences in this region play a key role in the ability of the virus to interact with hematopoietic cells in vivo and in vitro.     

Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis.

We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.     

Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.     

Temperature-sensitive mutants of Abelson murine leukemia virus deficient in protein tyrosine kinase activity.

The effect of two missense mutations in abl on transformation by Abelson murine leukemia virus was evaluated. These mutations led to the substitution of a histidine for Tyr-590 and a glycine for Lys-536. Both changes gave rise to strains that were temperature dependent for transformation of both NIH 3T3 cells and lymphoid cells when expressed in the context of a truncated Abelson protein. In the context of the prototype P120 v-abl protein, the Gly-536 substitution generated a host range mutant that induced conditional transformation in lymphoid cells but had only a subtle effect on NIH 3T3 cells. The combination of both substitutions gave rise to a P120 strain that was temperature sensitive for both NIH 3T3 and lymphoid cell transformation. The Abelson proteins encoded by the temperature-sensitive strain displayed in vitro kinase activities that were reduced when compared with those of wild-type proteins. In vivo, levels of phosphotyrosine were reduced only at the restrictive temperature. Analysis of cells expressing either the wild-type P160 v-abl protein or the P210 bcr/abl protein and an Abelson protein encoded by a temperature-sensitive strain failed to correct this defect, suggesting either that tyrosine phosphorylation in vivo is an intramolecular reaction or that the protein encoded by the temperature-sensitive strain is a poor substrate for tyrosine phosphorylation in vivo. These results raise the possibility that tyrosine phosphorylation of Abelson protein plays a role in transformation.     

SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.     

Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation.

Abelson murine leukemia virus transforms both lymphoid cells and fibroblasts in vitro and induces a unique type of thymus-dependent lymphoma in vivo. Four fibroblast-transforming strains of Abelson murine leukemia virus were identified, based on the sizes of the Abelson murine leukemia virus-specific phosphoproteins produced by these isolates. Two of these strains, the standard P120- and the P160-producing viruses, transformed lymphoid cells efficiently in vitro and induced Abelson disease in vivo. Two other strains, which synthesized small Abelson murine leukemia virus-specific proteins with molecular weights of 90,000 (P90) and 100,000 (P100), transformed lymphoid cells very poorly both in vitro and in vivo. The reduced oncogenic potentials of these isolates were correlated with a high level of synthesis of fairly unstable P90 and P100. In addition, neither P90 nor P100 functional efficiently in protein kinase assays. The correlation of abnormal metabolism and deficient protein kinase activity with the reduced oncogenic potentials of these virus strains supported a direct role for these proteins and the kinase activity in transformation. Furthermore, these results suggested that the requirements for lymphoid cell transformation and fibroblast transformation are different.     

Immunoglobulin synthesis by lymphoid cells transformed in vitro by Abelson murine leukemia virus.

The majority of cell lines derived by infection of murine bone marrow cells with Abelson murine leukemia virus (A-MuLV) synthesize a mu chain but no detectable light chain. Aside from this mu-only phenotype, lines that make only light chain, both chains or no immunoglobulin-related polypeptides have also been found. Two lines have been studied in detail: one that makes only mu chain and one that makes only kappa light chain. Synthesis of both polypeptides can be increased by modifying the culture conditions so as to decrease the growth rate of the cells. Although some kappa chain secretion was observed, neither secreted nor surface mu was detected. We suggest that the mu- only phenotype may be an early normal step in the pathway of B lymphocyte maturation.     

Abelson murine leukemia virus mutants with alterations in the virus-specific P120 molecule.

Abelson murine leukemia virus (A-MuLV) is a replication-defective virus that transforms both fibroblasts and hematopoietic cells in vitro. The virus encodes a 120,000-molecular-weight protein (P120) that is composed of Moloney murine leukemia virus-derived gag gene sequences and A-MuLV--specific sequences. This protein is the only A-MuLV--encoded protein that has been detected, and thus P120 is a candidate for the transforming protein of A-MuLV. We now report isolation and characterization of three new A-MuLV isolates that do not synthesize P120 but do produce analogous proteins of larger (160,000 molecular weight) and smaller (100,000 and 90,000 molecular weight) size. All of these A-MuLV isolates transform fibroblasts and lymphoid cells in vitro. Because the different A-MuLV proteins vary in the A-MuLV--specific region of the molecule, these variants may set a maximum limit on the size of the A-MuLV transforming protein.     

Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus

Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein-serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+-calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.     

A natural human retrovirus efficiently complements vectors based on murine leukemia virus

Background

Murine Leukemia Virus (MLV) is a rodent gammaretrovirus that serves as the backbone for common gene delivery tools designed for experimental and therapeutic applications. Recently, an infectious gammaretrovirus designated XMRV has been identified in prostate cancer patients. The similarity between the MLV and XMRV genomes suggests a possibility that the two viruses may interact when present in the same cell.

Methodology/Principal Findings

We tested the ability of XMRV to complement replication-deficient MLV vectors upon co-infection of cultured human cells. We observed that XMRV can facilitate the spread of these vectors from infected to uninfected cells. This functional complementation occurred without any gross rearrangements in the vector structure, and the co-infected cells produced as many as 10⁴ infectious vector particles per milliliter of culture medium.

Conclusions/Significance

The possibility of encountering a helper virus when delivering MLV-based vectors to human cells in vitro and in vivo needs to be considered to ensure the safety of such procedures.     

Differentiation of cloned populations of immature B cells after transformation with Abelson murine leukemia virus

The nature of the target cell for Abelson virus transformation and the effect of transformation on B cell differentiation were studied with six cloned lines of nontransformed immature B lymphocytes. Three clones were at the pre-B cell stage of maturation prior to A-MuLV infection; two were at the B cell stage, and one appeared to represent a stage prior to rearrangement of the mu heavy chain gene. All six cloned lines could be transformed by Abelson virus. Many of the transformants of the pre-B cell clones underwent kappa light chain gene rearrangement and expression following viral infection. Distinct light chain gene rearrangements were segregated by further subcloning these transformed lines. Abelson virus infection of one cloned cell line believed to represent a stage of maturation prior to the pre-B cell stage produced pre-B cell transformants with a variety of heavy chain gene rearrangements. Thus B lymphoid target cells for Abelson virus are not restricted to a single developmental stage, and some transformed subclones can undergo extensive immunoglobulin gene rearrangements shortly after viral infection.     

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

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