Ecotropic Moloney Murine Leukemia Virus Research Articles

The Nef-Like Effect of Murine Leukemia Virus Glycosylated Gag on HIV-1 Infectivity Is Mediated by Its Cytoplasmic Domain and Depends on the AP-2 Adaptor Complex

Human immunodeficiency virus type 1 (HIV-1) Nef enhances the infectivity of progeny virions. However, Nef is dispensable for the production of HIV-1 virions of optimal infectivity if the producer cells are superinfected with certain gammaretroviruses. In the case of the ecotropic Moloney murine leukemia virus (M-MLV), the Nef-like effect is mediated by the glycosylated Gag (glycoGag) protein. We now show that the N-terminal intracellular domain of the type II transmembrane protein glycoGag is responsible for its effect on HIV-1 infectivity. In the context of a fully active minimal M-MLV glycoGag construct, truncations of the cytoplasmic domain led to a near total loss of activity. Furthermore, the cytoplasmic domain of M-MLV glycoGag was fully sufficient to transfer the activity to an unrelated type II transmembrane protein. Although the intracellular region of glycoGag is relatively poorly conserved even among ecotropic and xenotropic MLVs, it was also fully sufficient for the rescue of nef-deficient HIV-1 when derived from a xenotropic virus. A mutagenic analysis showed that only a core region of the intracellular domain that exhibits at least some conservation between murine and feline leukemia viruses is crucial for activity. In particular, a conserved YXXL motif in the center of this core region was critical. In addition, expression of the μ2 subunit of the AP-2 adaptor complex in virus producer cells was essential for activity. We conclude that the ability to enhance HIV-1 infectivity is a conserved property of the MLV glycoGag cytoplasmic domain and involves AP-2-mediated endocytosis. The Nef protein of HIV-1 and the entirely unrelated glycosylated Gag (glycoGag) protein of a murine leukemia virus (MLV) similarly enhance the infectiousness of HIV-1 particles by an unknown mechanism. MLV glycoGag is an alternative version of the structural viral Gag protein with an extra upstream region that provides a cytosolic domain and a plasma membrane anchor. We now show for the first time that the cytosolic domain of MLV glycoGag contains all the information needed to enhance HIV-1 infectivity and that this function of the cytosolic domain is conserved despite limited sequence conservation. Within the cytosolic domain, a motif that resembles a cellular sorting signal is critical for activity. Furthermore, the enhancement of HIV-1 infectivity depends on an endocytic cellular protein that is known to interact with such sorting signals. Together, our findings implicate the endocytic machinery in the enhancement of HIV-1 infectivity by MLV glycoGag.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.

Effects of the ligand sequence modifications on the retargeted transduction by the retroviral vector having a ligand-chimeric Env protein

There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1alpha) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 10(4) c.f.u. ml(-1). Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 10(6) c.f.u. ml(-1). The SDF-1alpha ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3-51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.

Mechanisms underlying glycosylation-mediated loss of ecotropic receptor function in murine MDTF cells and implications for receptor evolution

A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus-host interactions.

Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein.

To develop a reliable strategy for cell-specific delivery of retroviral vectors, we genetically modified the envelope (Env) protein of the ecotropic Moloney murine leukemia virus. We found a site in the variable region A, where the insertion of ligands, epidermal growth factor (EGF) and stromal-derived factor-1 alpha (SDF-1 alpha), was possible without abolishing virion incorporation of the Env protein and its ecotropic entry function. The vector containing the EGF-Env did not show the EGF receptor-dependent transduction. The vector containing the SDF-1 alpha-Env, however, specifically transduced human cells expressing CXCR4, the receptor for SDF-1 alpha, at titers of 10(3)-10(4) c.f.u./ml. Further experiments showed that the CXCR4-dependent transduction was based on the specific interaction between the SDF-1 alpha moiety of the SDF-1 alpha-Env and CXCR4 and was independent of the ecotropic entry function. The direct targeting of the retroviral vector may be possible if the proper chimeric Env structure and the appropriate ligand-receptor system are employed.

A chimeric human T-cell lymphotropic virus type 1 with the envelope glycoprotein of Moloney murine leukemia virus is infectious for murine cells.

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.

Selective transduction of HIV-1-infected cells by the combination of HIV and MMLV vectors.

Human immunodeficiency virus 1 (HIV-1)-infected cells are important targets of gene therapy for acquired immune deficiency syndrome. We have developed a novel strategy for targeted gene transfer into HIV-1-infected cells based on 2-step gene transfer. The first step involves the stable introduction of the HIV vector containing the ecotropic Moloney murine leukemia virus (MMLV) receptor gene (EcoRec) into human CD4+ T cells as a molecular switch. Because the HIV-long terminal repeat (HIV-LTR) is Tat inducible, it is expected that EcoRec is expressed only after HIV-1 infection. Northern blot analysis and a retrovirus binding assay confirmed that the HIV-LTR of the integrated vector was silent in transduced cells but strongly transactivated in HIV-1 infection. High levels of EcoRec expression were observed only in HIV-1-infected cells. These cells became highly susceptible to ecotropic MMLV infection and, therefore, in the second step, HIV-1-infected cells were selectively transduced with ecotropic MMLV vectors. More than 70% of HIV-1-infected cells were transduced by this strategy. These findings indicate that this 2-step method can be used for selective and stable gene transfer into HIV-1-infected cells.

Functional characterization of the N termini of murine leukemia virus envelope proteins.

The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry.

Insertional mutagenesis of preneoplastic astrocytes by Moloney murine leukemia virus

Retroviral infection can induce transcriptional activation of genes flanking the sites of proviral integration in target cells. Because integration is essentially random, this phenomenon can be exploited for random mutagenesis of the genome, and analysis of integration sites in tumors may identify potential oncogenes. Here we have investigated this strategy in the context of astrocytoma progression. Neuroectodermal explants from astrocytoma-prone GFAP-v-src transgenic mice were infected with the ecotropic Moloney murine leukemia virus (Mo-MuLV). In situ hybridization and FACS analysis indicated that astrocytes from E12.5-13.5 embryos were highly susceptible to retroviral infection and expressed viral RNA and proteins both in vitro and in vivo. In average 80% of neuroectodermal cells were infected in vitro with 9-14 proviral integrations per cell. Virus mobility assays confirmed that Mo-MuLV remained transcriptionally active and replicating in neuroectodermal primary cultures even after 45 days of cultivation. Proviral insertion sites were investigated by inverse long-range PCR. Analysis of a limited number of provirus flanking sequences in clones originated from in vitro infected GFAP-v-src neuroectodermal cells identified loci of possible relevance to tumorigenesis. Therefore, the approach described here might be suitable for acceleration of tumorigenesis in preneoplastic astrocytes. We expect this method to be useful for identifying genes involved in astrocytoma development/progression in animal models.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

AbstractThe low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Role of the Cytoplasmic Tail of Ecotropic Moloney Murine Leukemia Virus Env Protein in Fusion Pore Formation

Fusion between cells expressing envelope protein (Env) of Moloney murine leukemia virus and target cells were studied by use of video fluorescence microscopy and electrical capacitance measurements. When the full-length 632-amino-acid residue Env was expressed, fusion did not occur at all for 3T3 cells as target and only somewhat for XC6 cells. Expression of Env 616*-a construct of Env with the last 16 amino acid residues (617 to 632; the R peptide) deleted from its C terminus to match the proteolytically cleaved Env produced during viral budding-resulted in high levels of fusion. Env 601*, lacking the entire cytoplasmic tail (CT) (identified by hydrophobicity), also led to fusion. Truncation of an additional six residues (Env 595*) abolished fusion. The kinetics of forming fusion pores did not depend on whether cells were first prebound at 4 degrees C and the time until fusion measured after the temperature was raised to 37 degrees C or whether cells were first brought into contact at 37 degrees C and the time until fusion immediately measured. This similarity in kinetics indicates that binding is accomplished quickly compared to subsequent steps in fusion. The fusion pores formed by Env 601* and Env 616* had the same initial size and enlarged in similar manners. Thus, once the R peptide is removed, the CT is not needed for fusion and does not affect formed pores. However, residues 595 to 601 are required for fusion. It is suggested here that the ectodomain and membrane-spanning domain of Env are directly responsible for fusion and that the R peptide affects their configurations at some point during the fusion process, thereby indirectly controlling fusion.

A Hydrophobic Patch in Ecotropic Murine Leukemia Virus Envelope Protein Is the Putative Binding Site for a Critical Tyrosine Residue on the Cellular Receptor

In the receptor for ecotropic murine leukemia viruses, tyrosine 235 contributes a critical hydrophobic side chain to the virus-receptor interaction (14). Here we report that tryptophan 142 in ecotropic Moloney murine leukemia virus envelope protein is essential to virus binding and infection. Replacement of tryptophan 142 by alanine or serine resulted in misfolding. However, replacement by methionine (W142M) allowed correct folding of the majority of glycoprotein molecules. W142M virus showed a marked reduction in virus binding and was almost noninfectious, suggesting that tryptophan 142 is involved in receptor binding. In contrast, W142Y virus containing a replacement of tryptophan 142 with an aromatic residue (tyrosine) was as efficient as wild-type virus in infection and binding of cells expressing the wild-type receptor. However, W142Y virus was 100-fold less efficient than wild-type virus in infection of cells expressing a mutant receptor containing tryptophan instead of the critical tyrosine. These results strongly support tryptophan 142 being an essential residue on the virus envelope protein that interacts directly with the critical hydrophobic residue at position 235 of the ecotropic receptor. Tryptophan 142 forms one side of a shallow hydrophobic pocket on the surface of the envelope protein, suggesting that it might comprise the complete putative binding site for tyrosine 235. We discuss the implications of our findings with respect to two models of the envelope protein trimer. Interestingly, both models place tryptophan 142 at the interface between adjacent subunits of the trimer.

Retrovirus Vectors Displaying the IgG-Binding Domain of Protein A

We have designed and constructed retrovirus particles displaying the IgG-binding domain of protein A. We fused the gene for the synthetic antibody-binding portion of protein A with the envelope gene of ecotropic Moloney murine leukemia virus. The fusion gene was coexpressed in ecotropic retroviral packaging cells, and retrovirus particles with IgG-binding activities were recovered. In principle, the protein A–envelope chimeric retrovirus complexed with specific monoclonal antibody could be used for cell-targeted gene delivery.

Variable Transduction Efficiency of Murine Leukemia Retroviral Vector on Mammalian Cells: Role of Cellular Glycosylation

To elucidate the cellular tropism of Moloney murine leukemia virus (MuLV), we have studied the transduction efficiency of a recombinant MuLV vector carrying the β-galactosidase reporter gene on a variety of rodent cells. Under optimal conditions forin vitrocell transduction, primary cultures of adult rat fibroblasts derived from various organs were very poorly transduced by the ecotropic MuLV vector (0.02–0.12%) when compared to immortalized cells such as the F2408 (42%) and 3Y1 (defined as 100%) lines. Primary cultures of fibroblasts from neonatal (3.7%) or embryonic rat tissues (4.6%) and primary cultures of rat mammary epithelial cells (3–4%) were somewhat more susceptible. Immortalization of rodent fibroblasts with Polyomavirus Large T, SV40 Large T, and E6-E7 genes of human papilloma virus resulted in a modest or minimal increase in transduction efficiency, and introduction of the transforming genes v-Src, v-Ras, and v-Raf was in most instances associated with a decrease in MuLV vector entry. Variability of transduction efficiency was not related to differences in cellular growth rate and treatment of MuLV vectorsin vitrowith deoxyribonucleoside triphosphates and treatment of cells in culture with protease inhibitors failed to modify cellular entry of the MuLV vector. On the other hand, inhibition of cellular glycosylation with swansonine, 1-deoxymannojirimycin and, primarily, tunicamycin enhanced entry of the ecotropic vector by up to 220-fold, particularly into cells which were otherwise highly resistant. These findings demonstrate major differences in transduction efficiency of the ecotropic MuLV vector on rodent cells and indicate that cellular glycosylation plays a critical role in determining MuLV cellular tropism.

Establishment and characterization of conditionally immortalized astrocytes to study their interaction with ts1, a neuropathogenic mutant of Moloney murine leukemia virus.

The cytopathic infection of primary astrocytes with ts1, a neuroimmunopathogenic mutant of Moloney murine leukemia virus (MuLV), has been correlated to intracellular accumulation of viral precursor envelope protein gPr80env. To further study this specific virus-astrocyte interaction in a homogenous population, several immortal astrocyte lines were established from neonatal FVB/N mice using the temperature-sensitive SV40 tsA58 T antigen. These cells expressed glial fibrillary acidic protein, vimentin and T antigen; appeared nontransformed; were star-shape with long processes. They were susceptible to ts1 infection and suffered a cytopathic effect similar to that caused by ts1 infection of primary astrocytes. This cytopathic effect was characterized by growth inhibition, loss of cell processes and syncytium formation. Some cells also rounded up, formed mini cells and became detached from the culture dish. As in primary astrocytes, the processing of gPr80env in the immortalized astrocytes was inefficient. Since the envelope proteins interact with the ecotropic MuLV receptor both intracellularly and on the cell surface and since the receptor has been shown to be an arginine transporter, we attempted to determine the effect of ts1 on arginine uptake by these cells. Our results showed that in both immortalized and primary astrocytes, ts1 infection reduced the uptake of arginine more than did wild-type virus infection. Since arginine localizes predominantly in astrocytes in the CNS and has diverse functions, the decrease of arginine uptake in ts1-infected astrocytes may alter the metabolism of these cells, leading to impairment of their functions.

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.

Ligand-directed retroviral targeting of human breast cancer cells.

We explored the feasibility of designing retroviral vectors that can target human breast cancer cells with characteristic receptors via ligand-receptor interaction. The ecotropic Moloney murine leukemia virus envelope was modified by insertion of sequences encoding human heregulin. Ecotropic virus, which normally does not infect human cells, when pseudotyped with the modified envelope protein now crosses species to infect human breast cancer cell lines that overexpress HER-2 (human epidermal growth factor receptor; also called ERBB2) and HER-4 (also called ERBB4), while human breast cancer cell lines expressing low levels of these receptors remain resistant to infection. Since about 20% of human breast cancers overexpress HER-2 and some of breast cancer cell lines overexpress both HER-2 and HER-4, cell-specific targeting of retroviral vectors may provide a different approach for in vivo gene therapy of this type of breast cancer.

Tissue-Specific Targeting of Retroviral Vectors Through Ligand-Receptor Interactions

The development of retroviral vectors that target specific cell types could have important implications for the design of gene therapy strategies. A chimeric protein containing the polypeptide hormone erythropoietin and part of the env protein of ecotropic Moloney murine leukemia virus was engineered into the virus. This murine virus became several times more infectious for murine cells bearing the erythropoietin receptor, and it also became infectious for human cells bearing the erythropoietin receptor. This type of tissue-specific targeting by means of ligand-receptor interactions may have broad applications to a variety of gene delivery systems.

Oncogenicity of human N-ras oncogene and proto-oncogene introduced into retroviral vectors

The N-ras gene is the only member of the ras family which has never been naturally transduced into a retrovirus. In order to study the in vitro and in vivo oncogenicity of N-ras and to compare its pathogenicity to that of H-ras, we have inserted an activated or a normal form of human N-ras cDNA into a slightly modified Harvey murine sarcoma virus-derived vector in which the H-ras p21 coding region had been deleted. The resulting constructions were transfected into NIH 3T3 cells. The activated N-ras-containing construct (HSN) induced 10(4) foci per microgram of DNA and was found to be as transforming as H-ras was. After infection of the transfected cells by either the ecotropic Moloney murine leukemia virus or the amphotropic 4070A helper viruses, rescued transforming viruses were injected into newborn mice. Both pseudotypes of HSN virus containing activated N-ras induced the typical Harvey disease with similar latency. However, we found that the virus which contained normal N-ras p21 (HSn) was also pathogenic and induced splenomegaly, lymphadenopathies, and sarcoma in mice after a latency of 3 to 7 weeks. In addition, Moloney murine leukemia virus pseudotypes of N-ras caused neurological disorders in 30% of the infected animals. These results differed markedly from those of previous experiments in which we had inserted the activated form of N-ras in the pSV(X) vector: the resulting SVN-ras virus was transforming on NIH 3T3 cells but was poorly oncogenic in vivo (M. Souyri, C. F. Koehne, P. V. O'Donnel, T. H. Aldrich, M. E. Furth, and E. Fleissner, Virology 158:69-78). However, similarly poor oncogenicity was also observed when the v-H-ras coding sequence was inserted in pSV(X) vector, which indicated that the vector sequences play a crucial role in the pathogenicity of a given oncogene. Altogether, these data demonstrated unequivocally that N-ras is potentially as oncogenic as H-ras and that such oncogenic effect could depend on the vector environment.