Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.     

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.     

Appearance of Mink Cell Focus-Inducing Recombinants during In Vivo Infection by Moloney Murine Leukemia Virus (M-MuLV) or the Mo+PyF101 M-MuLV Enhancer Variant: Implications for Sites of Generation and Roles in Leukemogenesis

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.     

The leukemogenic potential of an enhancer variant of Moloney murine leukemia virus varies with the route of inoculation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.     

Effects of nonleukemogenic and wild-type Moloney murine leukemia virus on lymphoid cells in vivo: identification of a preleukemic shift in thymocyte subpopulations.

Infection of mice with Moloney murine leukemia virus (M-MuLV) as well as with a nonpathogenic variant, Mo+PyF101 M-MuLV, was studied. Mo+PyF101 M-MuLV differs from wild-type M-MuLV by the addition of enhancer sequences from polyomavirus in the long terminal repeat. Previous experiments indicated that Mo+PyF101 establishes infection in animals, even though it does not induce disease. In vivo infection studies with particular attention to the thymus were performed, since the thymus is the target organ for M-MuLV leukemogenesis. Mice inoculated at birth with wild-type M-MuLV developed maximal levels of thymic infection by 2 to 3 weeks. Animals inoculated with Mo+PyF101 M-MuLV showed considerably less thymic infection at early times (2 to 4 weeks); nevertheless, by 5 to 6 weeks infection equivalent to wild-type M-MuLV-inoculated animals developed. Therefore the nonpathogenicity of Mo+PyF101 M-MuLV did not simply reflect a lack of thymotropism. Furthermore, thymic infection by itself may not be sufficient to induce leukemia. The relative deficit of Mo+PyF101 M-MuLV thymic infection at early versus late times did not reflect a change in the nature of the cells in the thymus, since in vitro infection of primary thymocytes from 2- and 6-week-old animals was equally efficient. One possible explanation is that infected thymocytes normally arise from progenitor cells which were infected in the bone marrow or spleen, and the cells restricted for Mo+PyF101 M-MuLV are located in those organs. Comparison of wild-type and Mo+PyF101 M-MuLV also allowed identification of important preleukemic changes in the thymus of wild-type M-MuLV-inoculated mice. Flow cytometry with monoclonal antibodies specific for thymocyte subpopulations was used. Staining of cells for Thy-1 or Thy-1.2 antigens indicated a shift toward low or negative cells. A concomitant increase in cells positive for antigen Pgp-1 was also observed. This is consistent with an increase in the relative frequency of immature blastlike cells. Importantly, thymuses from mice inoculated with Mo+PyF101 M-MuLV did not show these shifts in thymocyte subpopulations.     

Preleukemic hematopoietic hyperplasia induced by Moloney murine leukemia virus is an indirect consequence of viral infection.

We previously showed that neonatal mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibit a preleukemic state characterized by splenomegaly and increased numbers of hematopoietic progenitors. An M-MuLV variant with greatly reduced leukemogenic potential, Mo+PyF101 M-MuLV, does not generally induce this preleukemic state. In order to investigate the mechanism involved in M-MuLV induction of preleukemic hyperplasia, we tested the CFU-mixed myeloid and erythroid (CFUmix) from M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice for the presence of virus by antibody staining and for the release of infectious virus. The majority of CFUmix colonies from both M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice contained infectious virus even though M-MuLV-inoculated mice showed elevated levels of CFUmix while the Mo+PyF101 M-MuLV-inoculated mice did not. This indicates that direct infection of hematopoietic progenitors was not sufficient to induce hyperplasia. Rather, hematopoietic hyperplasia may result indirectly from infection of some other cell type.     

Detection of lymphoid leukemia colony-forming cells in abelson virus infected mice: Differences in inbred strains

BALB/c or DBA/2 mice were infected with Abelson murine leukemia virus (A-MuLV), pseudotype Molony murine leukemia virus (M-MuLV). Infection of these mice with 10⁴ focus-forming units of A-MuLV (M-MuLV) induced overt leukemia, detectable grossly or microscopically in 90% of the mice at 20–38 days. However, these methods did not detect leukemia at 17 days or before. Bone marrow cells from A-MuLV-infected leukemic or preleukemic mice were placed in tissue culture in a soft agarose gel. Cells from leukemic or preleukemic BALB/c mice grew to form colonies of 10³ cells or more, composed of lymphoblasts, whereas marrow cells from normal uninfected mice did not. Cells from these colonies grew to form ascitic tumors after intraperitoneal inoculation into pristane-primed BALB/c recipient. Colony-forming leukemia cells could be detected in the marrow of A-MuLV-infected mice as early as 8 days after virus incoluation. The number of colony-forming leukemia cells increased as a function of time after virus inoculation. Colony-forming leukemia cells require other cells in order to replicate in tissue culture. Normal bone marrow cells, untreated or after treatment with mitomycin-C, provide this “helper” function. Only in the presence of untreated or mitomycin-C treated helper cells was the number of colonies approximately proportional to the number of leukemia cells plated. Marrow cells from leukemic BALB/c mice form more colonies than those from leukemic DBA/2 mice. The number of colonies formed per 10³ microscopically identifiable leukemia cells plated was determined to be 2–3 for leukemic BALB/c mice and 0.3 for DBA/2 mice. Cocultivation of leukemic DBA/2 marrow cells with mitomycin-C treated normal BALB/c cells did not increase the number of colonies formed by the DBA/2 leukemic cells. Thus, the decreased ability of DBA/2 leukemia cells to form colonies appears to be a property of the leukemia cell population.     

Moloney Murine Leukemia Virus Infects Cells of the Developing Hair Follicle after Neonatal Subcutaneous Inoculation in Mice

The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo+PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo+PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo+PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-M-MuLV CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo+PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo+PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLV-based vector expressing β-galactosidase also infected these cells after a s.c. inoculation.     

A multistep process of leukemogenesis in Moloney murine leukemia virus-infected mice that is modulated by retroviral pseudotyping and interference.

Mixed retroviral infections frequently exhibit pseudotyping, in which the genome of one virus is packaged in a virion containing SU proteins encoded by another virus. Infection of mice by Moloney murine leukemia virus (M-MuLV), which induces lymphocytic leukemia, results in a mixed viral infection composed of the inoculated ecotropic M-MuLV and polytropic MuLVs generated by recombination of M-MuLV with endogenous retroviral sequences. In this report, we describe pseudotyping which occurred among the polytropic and ecotropic MuLVs in M-MuLV-infected mice. Infectious center assays of polytropic MuLVs released from splenocytes or thymocytes of infected mice revealed that polytropic MuLVs were extensively pseudotyped within ecotropic virions. Late in the preleukemic stage, a dramatic change in the extent of pseudotyping occurred in thymuses. Starting at about 5 weeks, there was an abrupt increase in the number of thymocytes that released nonpseudotyped polytropic viruses. A parallel increase in thymocytes that released ecotropic M-MuLV packaged within polytropic virions was also observed. Analyses of the clonality of preleukemic thymuses and thymomas suggested that the change in pseudotyping characteristics was not the result of the emergence of tumor cells. Examination of mice infected with M-MuLV, Friend erythroleukemia virus, and a Friend erythroleukemia virus-M-MuLV chimeric virus suggested that the appearance of polytropic virions late in the preleukemic stage correlated with the induction of lymphocytic leukemia. We discuss different ways in which pseudotypic mixing may facilitate leukemogenesis, including a model in which the kinetics of thymic infection, modulated by pseudotyping and viral interference, facilitates a stepwise mechanism of leukemogenesis.     

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.     

Moloney Murine Leukemia Virus-Induced Preleukemic Thymic Atrophy and Enhanced Thymocyte Apoptosis Correlate with Disease Pathogenicity

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks postinoculation) a marked decrease in thymic size is apparent for M-MuLV-inoculated mice in comparison to age-matched uninoculated mice. We were interested in studying whether the thymic regression was due to an increased rate of thymocyte apoptosis in the thymi of M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated subcutaneously (s.c.) with wild-type M-MuLV (approximately 10⁵ XC PFU). Mice were sacrificed at 4 to 11 weeks postinoculation. Thymic single-cell suspensions were prepared and tested for apoptosis by two-parameter flow cytometry. Indications of apoptosis included changes in cell size and staining with 7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis were significantly higher in M-MuLV-inoculated mice than in uninoculated control animals, and the levels of apoptosis were correlated with thymic atrophy. To test the relevance of enhanced thymocyte apoptosis to leukemogenesis, mice were inoculated with the Mo+PyF101 enhancer variant of M-MuLV. When inoculated intraperitoneally, a route that results in wild-type M-MuLV leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis comparable to those seen with wild-type M-MuLV. However, in mice inoculated s.c., a route that results in attenuated leukemogenesis, significantly lower levels of apoptosis were observed. This supported a role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis. To examine the possible role of mink cell focus-forming (MCF) recombinant virus in raising levels of thymocyte apoptosis, MCF-specific focal immunofluorescence assays were performed on thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101 M-MuLV. The results indicated that infection of thymocytes by MCF virus recombinants is not required for the increased level of apoptosis and thymic atrophy.     

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.     

Generation and characterization of a recombinant Moloney murine leukemia virus containing the v-myc oncogene of avian MC29 virus: in vitro transformation and in vivo pathogenesis.

A new retrovirus consisting of the v-myc oncogene sequences of avian MC29 virus inserted into the genome of Moloney murine leukemia virus (M-MuLV) was generated. This was accomplished by constructing a recombinant DNA clone containing the desired organization, introducing the recombinant DNA into mouse NIH 3T3 cells, and superinfecting the cells with replication-competent M-MuLV. The construction was designed so that an M-MuLV gag-myc fusion protein would be produced. The resulting virus, M-MuLV(myc), morphologically transformed uninfected NIH 3T3 cells. Stocks of M-MuLV(myc)-M-MuLV were infected into secondary mouse embryo cultures. M-MuLV(myc) induced striking growth and proliferation of hematopoietic cells. These cells were of the myeloid lineage by morphology, phagocytic properties, and surface staining with Mac-1 and Mac-2 monoclonal antibodies. They resembled mature macrophages, although they displayed minor properties of immaturity. The myeloid cells were transformed in comparison with uninfected myeloid cells since they were less adherent and had unlimited proliferative capacity and reduced growth factor requirements. The transformed myeloid cells with proliferative potential were actually myeloid progenitors which apparently underwent terminal differentiation to macrophages. It was possible to derive a permanent line of factor-independent macrophages from M-MuLV(myc)-transformed myeloid cells. M-MuLV(myc) also immortalized and morphologically transformed mouse embryo fibroblasts. These in vitro properties closely resembled the biological activity of MC29 virus in avian cells and suggested that the nature of the v-myc oncogene was an important determinant in transformation specificity. Neonatal NIH Swiss mice inoculated intraperitoneally with M-MuLV(myc)-M-MuLV only developed lymphoblastic lymphoma characteristic of the M-MuLV helper alone, and no acute fibrosarcomas or myeloid tumors resulted. In light of the strong myeloid transformation observed in vitro, the absence of acute in vivo myeloid disease was noteworthy. Interestingly, when a derivative of M-MuLV(myc) carried by a nonpathogenic amphotropic MuLV helper was inoculated, T lymphomas developed with long latency. Molecular hybridization confirmed that these tumors contained M-MuLV(myc).     

Mink cell focus-forming murine leukemia virus killing of mink cells involves apoptosis and superinfection

Induction of apoptosis by different types of pathogenic retroviruses is an important step in disease development. We have observed that infection of thymic lymphocytes by the mink cell focus-forming murine leukemia virus (MCF MLV) during the preleukemic period resulted in an enhancement of apoptosis of these cells. To further study the ability of MCF MLVs to induce apoptosis and the role of this process in viral pathogenesis, we have developed an in vitro system of virus-induced apoptosis. MCF13 MLV infection of mink epithelial cells resulted in the production of cytopathic foci. In contrast, infection of mink cells with the 4070A amphotropic MLV did not produce any cytopathic effects. Staining of MCF13 MLV-infected cells with propidium iodide and annexin V-fluorescein isothiocyanate indicated that virus-induced cell death was due to apoptosis. At 6 days postinfection, the percentage of apoptotic MCF13 MLV-infected cells was 27% compared with 2 to 3% for mock- or amphotropic MLV-infected cells, representing a 9- to 14-fold difference. Assays for caspase-3 activation confirmed the detection by flow cytometry of apoptosis of MCF13 MLV-infected cells. Large amounts of unintegrated linear viral DNA were detectable by Southern blot analysis during the acute phase of infection, which indicated that MCF13 MLV is able to superinfect mink cells. Unintegrated viral DNA of only the linear form was detectable in thymic lymphocytes isolated from MCF13 MLV-inoculated mice during the preleukemic period. These results indicated that the ability of MCF13 MLV to induce apoptosis is correlated with its ability to superinfect cells and that this occurs as an early step in thymic lymphoma development.     

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.     

Simian immunodeficiency virus inhibits bone marrow hematopoietic progenitor cell growth.

The pathogenic mechanisms underlying the depressed hematopoietic functions seen in human immunodeficiency virus-infected individuals were explored in rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac). Bone marrow hematopoietic progenitor cell colony formation, both granulocyte/macrophage (CFU-GM) and erythrocyte (BFU-E), was shown to be decreased in number in SIVmac-infected rhesus monkeys. SIVmac was readily isolated from bone marrow cells of infected monkeys and was shown to be harbored in macrophages rather than T lymphocytes. The in vitro infection of normal bone marrow cells by SIVmac inhibited colony formation. A striking in vivo correlation between increased SIVmac load in bone marrow cells and decreased hematopoietic progenitor cell colony growth was also shown. Finally, inhibition of SIVmac replication in bone marrow macrophages resulted in increased progenitor cell colony growth from bone marrow cells. These results suggest that the infection of bone marrow macrophages by the acquired immunodeficiency syndrome (AIDS) virus may contribute to depressed bone marrow hematopoietic progenitor cell growth. Moreover, inhibition of AIDS virus replication in these macrophages might induce significant improvement in hematopoietic function.     

T lymphocyte tolerance and early appearance of virus-induced cell surface antigens in Moloney-murine leukemia virus neonatally injected mice

Regression of Moloney-murine sarcoma virus- (M-MSV) induced sarcomas in normal adult mice is accompanied by generation of virus-specific cytotoxic T lymphocytes (CTL). However, when neonatal mice that were injected with Moloney-murine leukemia virus (M-MuLV carrier) were subsequently challenged as adults with M-MSV, the sarcomas did not regress nor did they generate CTL. This failure to produce CTL cannot be ascribed to nonspecific immunodepressive effects or to suppressor cell generation since M-MuLV carrier mice exhibit normal reactivity after allogeneic cell stimulation. Moreover, addition of M-MuLV-infected cells as the third party to cultures does not reduce activity of CTL from M-MSV immune mice. Since M-MSV and M-MuLV possess common antigens, the observed unresponsiveness was considered in relationship to induction of a T lymphocyte tolerance, which may follow introduction of foreign antigens at an early stage of development. In fact, it was observed that as early as 10 days after injection, thymus, lymph node, and spleen from M-MuLV carrier mice express virus-induced cell-surface antigens that not only are targets for M-MSV-immune CTL, but also induce in vitro a strong specific cytotoxic response. In addition, a cold target inhibition assay disclosed that the same antigens are shared by both M-MuLV infected and leukemia cells, even though they are less expressed on the surface of the former. The finding that the cytotoxicity of alloreactive lymphocytes from M-MuLV carrier mice is reduced after preincubation with M-MSV immune CTL confirms that virus infection does not bring about functional inactivation of lymphocytes. Finally, it was observed that virus antigen presence on lymphocytes from M-MuLV neonatally injected mice is closely related to subsequent leukemia development.     

Structure and function of bone marrow environment

Bone marrow and spleen are the major hematopoietic tissue in adult mice. However, little is known about the specific mechanism regulating hematopoiesis within these tissues. Since Dexter et al. first described conditions to maintain bone marrow hematopoiesis, long term bone marrow culture (LTBMC) has been developed in order to analyze the mechanism of the maintenance of proliferation and differentiation of hematopoietic stem cells in vitro. Furthermore, several stromal cell lines which are able to support the growth and differentiation of hematopoietic lineage, has been established from LTBMC. Although it is well known that bone marrow stromal cell lines are able to produce colony stimulating factors, it has been suggested that the stromal cell factors which involve membrane bound moieties must have a key role in the regulation of hematopoiesis. We expect that monoclonal antibodies to the surface of bone marrow stromal cells could detect such a critical stroma-associated protein that bounds the cell surface of the bone marrow stroma.     

Low-multiplicity infection of Moloney murine leukemia virus in mouse cells: effect on number of viral DNA copies and virus production in producer cells.

Mouse cells infected with Moloney murine leukemia virus (M-MuLV) were prepared by two methods, and the number of M-MuLV-specific DNA copies in the infected cells was measured. The number of M-MuLV-specific DNA copies detected varied from one to eight per infected cell in different cell lines. Cells in which multiple rounds of viral infection occurred during establishment had on the average more viral DNA copies than cells in which infection at low multiplicity was performed, followed by cloning of the cells. However, even in cells derived by the low multiplicity of infection method, most cell lines carried more than one copy of M-MuLV-specific DNA. Virus production per cell was also measured, and no strict correlation was observed between the number of M-MuLV DNA copies present and the amount of virus produced.