Harvey Murine Sarcoma Virus Research Articles

Deregulation of methionine metabolism as determinant of progression and prognosis of hepatocellular carcinoma.

The under-regulation of liver-specific MAT1A gene codifying for S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and the up-regulation of widely expressed MAT2A, MATII isozyme occurs in hepatocellular carcinoma (HCC). MATα1:MATα2 switch strongly contributes to the fall in SAM liver content both in rodent and human liver carcinogenesis. SAM administration to carcinogen-treated animals inhibits hepatocarcinogenesis. The opposite occurs in Mat1a-KO mice, in which chronic SAM deficiency is followed by HCC development. This review focuses upon the changes, induced by the MATα1:MATα2 switch, involved in HCC development. In association with MATα1:MATα2 switch there occurs, in HCC, global DNA hypomethylation, decline of DNA repair, genomic instability, and deregulation of different signaling pathways such as overexpression of c-MYC (avian myelocytomatosis viral oncogene homolog), increase of polyamine (PA) synthesis and RAS/ERK (Harvey murine sarcoma virus oncogene homolog/extracellular signal-regulated kinase), IKK/NF-kB (I-k kinase beta/nuclear factor kB), PI3K/AKT, and LKB1/AMPK axes. Furthermore, a decrease in MATα1 expression and SAM level induces HCC cell proliferation and survival. SAM treatment in vivo and enforced MATα1 overexpression or MATα2 inhibition, in cultured HCC cells, prevent these changes. A negative correlation of MATα1:MATα2 and MATI/III:MATII ratios with cell proliferation and genomic instability and a positive correlation with apoptosis and global DNA methylation are present in human HCC. Altogether, these data suggest that the decrease of SAM level and the deregulation of MATs are potential therapeutic targets for HCC.

Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity

A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Psi) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.

New Aspects on the Safety of Multidrug-Resistance 1 Gene Transfers: No Indication for Clonal Dominance after Long-Term Follow-Up in a Primate Transplantation Model.

It is of concern whether the introduction of a transgene into hematopoietic stem cells by retroviral vectors will lead to an alteration of the growth and engraftment characteristics. Earlier studies in mice indicated that retroviral multidrug-resistance 1 gene transfer may be associated with a myeloproliferative disorder. In human or primate cells this could not be reproduced in bulk cell populations. Analysis on the clonal level were lacking.One method to study the in vivo behaviour of repopulating progenitor and stem cells is marking the cells with replication-incompetent retroviral vectors that integrate into identifiable host DNA sequences, thus allowing the tracking of cell progeny based on unique proviral insertion sites.In this study CD34-enriched peripheral blood stem cells from 2 rhesus macaque monkeys were split into two aliquots and transduced either with a multidrug-resistance 1 gene-retroviral vector based on the Harvey murine sarcoma virus (HaMDR1-vector) or a NeoR-retroviral vector based on the Moloney murine leukaemia virus (G1Na-vector). After autologous retransplantation, DNA from blood and bone marrow was collected at different time points in a period of 4 years and the animals are still alive.By using a highly sensitive and specific ligation-mediated polymerase chain reaction (LM-PCR) followed by sequencing of vector integration sites, we found in animal M120 32 different contributing hematopoietic clones 8 weeks and 50 weeks after transplantation and in animal M038 17 clones 58 weeks after transplantation. Based on the difference between the sequences of the HaMDR1-LTR and the G1Na-LTR, the clones can be allocated definitely to one of the two vectors. Remarkably, 36 clones descend from the G1Na-vector, whereas only 13 clones descend from the HaMDR1-vector.We conclude that hematopoiesis in these monkeys is polyclonal for prolonged periods after transplantation and that MDR1 gene transfer does not confer a proliferative advantage over vector-control-transduced hematopoietic stem cells.

Long-term silencing of retroviral vectors is resistant to reversal by trichostatin A and 5-azacytidine.

One problem limiting the development of long-term gene replacement therapy is gene silencing. A variety of experiments have implicated DNA methylation and histone deacetylation in gene silencing and shown that the agents 5-azacytidine (5-Aza) and trichostatin A (TSA) are able to reverse these effects. To begin to investigate clinically relevant strategies to reverse silencing with these drugs, we transduced the MEL and FDCP-1 hematopoietic cell lines with Moloney murine leukemia virus (MMLV) and Harvey murine sarcoma virus (HMSV)-based retroviral vectors carrying the beta-galactosidase/neomycin resistance fusion gene (beta-geo). Fifty-one clones were isolated under G418 selection over 2 weeks and then allowed to grow without selection as beta-gal activity was monitored over time. More than 80% of these clones showed significant silencing over a period of 70-80 days. The clones were then exposed to a wide range of 5-Aza and TSA concentrations, both alone and in combination, in an effort to reverse silencing. Despite demonstration that the agents were able to decrease DNA methylation and increase histone acetylation, significant reversal of long-term silencing was not seen under any experimental condition. These results suggest that long-term retroviral silencing involves mechanisms in addition to DNA methylation and histone acetylation and that new pharmacologic strategies are needed to overcome the silencing process.

Identification of an internal ribosome entry segment in the 5' region of the mouse VL30 retrotransposon and its use in the development of retroviral vectors.

Mouse virus-like 30S RNAs (VL30m) constitute a family of retrotransposons, present at 100 to 200 copies, dispersed in the mouse genome. They display little sequence homology to Moloney murine leukemia virus (MoMLV), do not encode virus-like proteins, and have not been implicated in retroviral carcinogenesis. However, VL30 RNAs are efficiently packaged into MLV particles that are propagated in cell culture. In this study, we addressed whether the 5' region of VL30m could replace the 5' leader of MoMLV functionally in a recombinant vector construct. Our data confirm that the putative packaging sequence of VL30 is located within the 5' region (nucleotides 362 to 1149 with respect to the cap structure) and that it can replace the packaging sequence of MoMLV. We also show that VL30m contains an internal ribosome entry segment (IRES) in the 5' region, as do MoMLV, Friend murine leukemia virus, Harvey murine sarcoma virus, and avian reticuloendotheliosis virus type A. Our data show that both the packaging and IRES functions of the 5' region of VL30m RNA can be efficiently used to develop retrotransposon-based vectors.

Detection of a rare point mutation at codon 59 and relatively high incidence of H-ras mutation in Indian oral cancer.

Analysis of H-, K- and N-ras genes for point mutations by PCR-SSCP and direct sequencing of 46 oral SCCs that were previously analyzed for p53 mutations revealed that 9 (20%) had point mutations in either the H-ras or the N-ras. A novel mutation at codon 59 (GCC-ACC) of H-ras thus far reported only in v-H-ras of Harvey murine sarcoma virus was observed in a tumor of the cheek. Majority (8/9) of these mutations were observed in H-ras, one in N-ras and none in K-ras. This study indicated that the ras gene mutation was relatively high in oral cancers associated with tobacco chewing and the ras and p53 mutational events seem to be independent and mutually exclusive.

Construction and characterization of single-transcript tricistronic retroviral vectors using two internal ribosome entry sites.

We describe a series of retroviral vectors containing two internal ribosome entry sites (IRES) for the co-transcription of three genes. Transcription of the single-transcript tricistronic mRNA is under the control of a Harvey murine sarcoma virus long terminal repeat. The 5'-most open reading frame is under either cap-dependent or cap-independent translational control, while the two downstream open reading frames are translated in a cap-independent fashion using the initiation codons of their respective IRES elements. Both IRES elements are taken from the encephalomyocarditis virus. To characterize these vectors, we used the human multidrug resistance gene (MDR1) in the 5' position, the gene for green fluorescent protein (GFP) in the middle position, and neo in the 3' position. The vectors were either transfected directly into NIH3T3 mouse fibroblasts or packaged into retrovirus and then transduced into NIH3T3 cells. Gene transfer was followed by selection with colchicine, which selects for expression of the MDR1 gene, or with G418, which selects for expression of the neo gene. Thus, we could determine the function of the tricistronic vectors under conditions of selection for either the 5'-most or the 3'-most gene. In DNA-mediated transfections, we were able to achieve expression of all three open reading frames under either selection condition. We obtained higher expression of all three genes when colchicine was used to select for MDR1 expression than when G418 was used to select for neo expression. Expression of the non-selected GFP gene (the middle cistron) was unstable, most likely due to loss of integrated GFP DNA sequences during long-term culturing. We were able to achieve retrovirus-mediated transduction of all three genes, but this was an inefficient process.

High-Dose Multidrug Resistance in Primary Human Hematopoietic Progenitor Cells Transduced With Optimized Retroviral Vectors

Retroviral transfer of the multidrug-resistance 1 (mdr1) cDNA into primary human hematopoietic progenitor cells (HPC) of cancer patients undergoing high-dose chemotherapy has been proposed to protect the bone marrow from the dose-limiting cytotoxicity of cytostatic agents. Preclinical studies performed with vectors derived from the Moloney murine leukemia virus (MoMuLV) or the related Harvey murine sarcoma virus have established that chemoprotection of HPC is feasible. The efficacy of vector-mediated multidrug-resistance under high doses of cytostatic agents, however, remained unclear. We report here that this goal can only be achieved with improved vector design. Novel vectors termed SF-MDR and MP-MDR, which are based on the spleen focus-forming virus or the myeloproliferative sarcoma virus for the enhancer and the murine embryonic stem cell virus for the leader, significantly elevate survival of transduced primary human HPC under moderate doses of colchicine and paclitaxel in vitro when compared with a conventional MoMuLV-based vector. Importantly, SF-MDR and also MP-MDR confer an absolute advantage at high doses of paclitaxel in vitro corresponding to peak plasma levels achieved in patients during chemotherapy. This observation has important consequences for a variety of ongoing and planned gene therapy trials.

Expression of the Human Multidrug Resistance and Glucocerebrosidase cDNAs from Adeno-Associated Vectors: Efficient Promoter Activity of AAV Sequences andIn VivoDelivery via Liposomes

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

Bicistronic and Two-Gene Retroviral Vectors for UsingMDR1 as a Selectable Marker and a Therapeutic Gene

We describe a series of two-gene and bicistronic retroviral vectors that use the humanMDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we usedneoas a reporter gene for foreign gene expression and as an independently selectable marker for comparison withMDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. UsingMDR1–neovirus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting thatMDR1 is a more stringent selectable marker thanneoin NIH 3T3 and KB-3-1 cell lines. Whereasneogene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of theMDR1 gene product than did selection with G418, suggesting that the stringentMDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carryingMDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility forin vitroandin vivoapplications of gene transfer technology, including gene therapy.

Transformation of NIH 3T3 Cells by HER3 or HER4 Receptors Requires the Presence of HER1 or HER2

Members of the epidermal growth factor receptor (EGFR) subfamily of receptor protein tyrosine kinases have been implicated in the pathogenesis of various malignancies. The ability of one EGFR subfamily member to influence, or function synergistically with, another is likely to be a general feature of these receptors. To assess the role of receptor heterodimerization, we analyzed the ability of Neu differentiation factor (NDF) to induce cell growth and transformation of NIH 3T3 cells transfected with different combinations of the EGFR subfamily of receptors. NDF induced mitogenesis, but not transformation, of cells expressing either HER3 or HER4 alone. However, NDF-induced cell transformation was observed when either HER1 or HER2 was coexpressed with HER3 or HER4. In analogous receptor phosphorylation experiments, NDF-induced transphosphorylation appears to be correlated with synergistic transformation of NIH 3T3 cells. Interestingly, transphosphorylation between HER1 and HER4 can be stimulated by either EGF or NDF.

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

Retroviral coexpression of a multidrug resistance gene (MDR1) and human alpha-galactosidase A for gene therapy of Fabry disease.

Human alpha-galactosidase A (alpha-Gal A; EC.3.2.1.22) is a lysosomal exoglycosidase encoded by a gene on Xq22. Deficiencies of this enzyme result in Fabry disease, an X-chromosome-linked recessive disorder that leads to premature death in affected males. For treatment of genetic diseases, we have developed a retroviral vector system, pSXLC/pHa, that enables coexpression of drug-selectable markers with a second nonselectable gene as part of a bicistronic message using the promoter from the Harvey murine sarcoma virus and an internal ribosomal entry site (IRES) from encephalomyocarditis virus. Retroviral vectors based on this system that carry the human alpha-Gal A cDNA either upstream (pHa-alpha Gal-IRES-MDR) or downstream (pHa-MDR-IRES-alpha Gal) from the IRES relative to the drug-selectable MDR1 (P-glycoprotein) cDNA were constructed. Each of eight independent vincristine-resistant, pHa-alpha Gal-IRES-MDR-transfected clones and all four vincristine-resistant, pHa-alpha Gal-IRES-MDR retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. More than 50% of the vincristine-resistant, pHa-MDR-IRES-alpha Gal-transfected clones and all four vincristine-resistant, pHa-MDR-IRES-alpha Gal retrovirus-transduced clones showed significantly higher activity of alpha-Gal A than the parental cells. In these bicistronic vectors, the cDNA whose translation was cap-dependent (upstream) was expressed at higher levels than when the same cDNA was translated in an IRES-dependent manner (downstream). These vectors may prove useful in the gene therapy of Fabry disease.

Harvey Murine Sarcoma Virus/MDR1 Retrovital Vectors: Efficient Virus Production and Foreign Gene Transduction Using MDR1 as a Selectable Marker

Retroviruses are used for a variety of applications requiring the delivery of exogenous genes to cells and animals. For many of these applications, including gene therapy, safer and more efficient retroviral vectors are needed. Vectors based on Harvey murine sarcoma virus (HaMSV) are attractive because nearly all their viral sequences outside of the LTRs are derived from rat endogenous VL30 retroviruses. These sequences are not homologous to the functional viral mRNAs in commonly used retrovirus packaging cell lines, the packaging and dimerization domains of HaMSV are small and contain no splice donor sites, and the 5′ sequences of HaMSV appear to confer efficient packaging and stability on genomic RNAs. HaMSV/MDR1 vectors use the human multidrug resistance gene as a dominant, selectable, amplifiable marker for gene delivery, but current versions of these vectors are large, with over 3300 nt of HaMSV sequences downstream of MDR1. We analyzed the requirement for these downstream sequences in HaMSV vectors and found that modified HaMSV/MDR1 vectors lacking virtually all viral sequences downstream of MDR1 support the production of high-titer retroviruses and the efficient transduction, selection, and amplification of MDR1. A reduced-size HaMSV/MDR1 vector was further modified to include a second heterologous gene under the control of an internal SV40 promoter. Using MDR 1 as a selectable marker, we obtained efficient virus production, gene transduction, and expression of MDR1 plus the heterologous gene.

Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site.

We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.

Analytical Study of Rat Retrotransposon VL30 RNA Dimerization in Vitro and Packaging in Murine Leukemia Virus

A sequence of the rat retrotransposon virus-like 30 S RNA (VL30) located next to the 5′ end of the Harvey murine sarcoma virus (HaMSV) genome was recently found to form stable dimeric RNA in vitro and to direct the efficient packaging of VL30-derived recombinant RNAs into MuLV virions. To study the structure—function relationships of the rat VL30 dimerization—encapsidation signal (E/DLS), we have performed biochemical and genetic studies of rat VL30 RNA dimerization in vitro. The results show that temperature and specific cation/RNA interactions are important for VL30 dimerization in vitro. VL30 RNA dimerization is optimal at 55°C and Li+ dramatically enhances the stability of VL30 dimeric RNA. In addition, a genetic analysis of VL30 RNA dimerization reveals that a 5′ G-rich sequence is critical for dimer formation and that a UGUCUUGUC repeat contributes to VL30 dimer stability. Interesting enough, substitution of an A for G in the 5′ G-rich sequence is sufficient to abolish VL30 RNA dimerization in vitro. Taken together, these biochemical and genetic data indicate that dimerization of VL30 RNA involves non-canonical base-pairings and possibly purine—purine interactions. Nucleocapsid protein NCp10 of murine leukemia virus (MuLV), a gag-encoded protein that is tightly associated with genomic RNA in the virion core, has been shown to have nucleic acid binding and annealing activities. Here we report that the viral NCp10 protein is able to bind tightly to the retrotransposon VL30 RNA and to activate its dimerization. Moreover, mutations in the 5′ G-rich sequence of the VL30 dimerization sequence impaired NCp10 binding to RNA. Recombinant MLV-VL30 vectors with mutations in the VL30 dimerization sequence were constructed. Results obtained in vivo clearly show that the mutations that had a deleterious effect on the packaging of MLV-VL30 retroviral vectors in vivo were those that impaired VL30 RNA dimerization and interactions with NCp10 in vitro, even the single mutation in the 5′ G-rich region. Therefore, these findings suggest that packaging of VL30 RNA into MuLV virions requires specific interactions between RNA dimerization sequences and viral NC protein molecules.

Retroviral vectors containing chimeric promoter/enhancer elements exhibit cell-type-specific gene expression.

Retroviral vectors were constructed in which the U3 promoter/enhancer of Moloney murine leukemia (Mo-MLV) was replaced by the corresponding region from five related murine retroviruses--AKR murine leukemia virus (AKV), Harvey murine sarcoma virus (HaMSV), myeloproliferative sarcoma virus (MPSV), SL3-3, and the NZB-xenotropic virus (Xeno). In these vectors the chimeric long terminal repeat (chLTR) drives the expression of the chloramphenicol acetyl transferase (CAT) reporter gene that is followed by an internal SV40 virus early region promoter linked to the neomycin phosphotransferase II (NEO) gene. As an initial measure of the relative promoter/enhancer strength of the chLTR vectors, the murine NIH-3T3 cell line and the human JURKAT cell lines were transfected and assayed for CAT reporter activity. Relative to the MoMLV vector, the HaMSV construct was the most active in NIH-3T3 cells whereas the SL3-3 vector displayed the greatest activity in JURKAT cells. Retroviral vector producer cell populations and cell clones were established for each chLTR vector, and all were capable of yielding high vector titers (> 10(5) G418R cfu/ml on NIH-3T3). Supernatant from these cells was used to transduce both mouse and human cell lines and primary cells. In NIH-3T3 cells and two murine fibrosarcoma cell lines, the HaMSV chLTR vector was slightly more active than the MoMLV chLTR vector. In the human HepG2 and HeLa cell lines, the MPSV chLTR vector was the most active. Data from the human JURKAT T-cell line and a T cell line derived from an ADA-deficient severe combined immunodeficiency (SCID) patient demonstrate that the SL3-3 chLTR is the most active in these lymphoid cell lines. The greatest difference in the comparison of the different chLTR vectors was observed in primary human umbilical vein endothelial cells, where the MoMLV vector produced up to 100 times more CAT activity than the SL3-3 vector. These data suggest that the use of specific promoter/enhancer elements may lead to higher levels of gene expression following retroviral-mediated gene transfer into specific cell types and these observations may be useful in the design of human gene therapy experiments.

Drug-selected coexpression of human glucocerebrosidase and P-glycoprotein using a bicistronic vector.

Bicistronic cassettes under control of a single promoter have recently been suggested as useful tools for coordinate expression of two different foreign proteins in mammalian cells. Using the long 5' untranslated region of encephalomyocarditis virus as translational enhancer of the second gene, a bicistronic unit composed of cDNA for human P-glycoprotein [the product of the multidrug resistance gene, MDR1 (also called PGY1)] as selectable marker and cDNA for human glucocerebrosidase (GC; EC 3.2.1.45) (a membrane-associated lysosomal hydrolase) was constructed. NIH 3T3 cells transfected with a Harvey murine sarcoma virus retroviral vector carrying this bicistronic cassette (pHaMCG) express active P-glycoprotein and GC and expression of both proteins augments coordinately with selection for increased colchicine resistance. Percoll gradient analysis of homogenates showed that GC was targeted to the lysosomal fraction. The ability to select for expression of GC with natural product drugs after introduction of the pHaMCG retroviral vector may be useful in gene therapy strategies for Gaucher disease.

8 Two new (newborn) mouse models - harvey murine sarcoma virus (MSV-Har) and rauscher murine leukaemia virus (RLV), for anti retroviral drug testing: J.J. Harvey, M. Botcherby. Antigen Presentation Research Group, MRC Clinical Research Centre, Northwick Park, Harrow, U.K.

A structural protein with a molecular weight of approximately 12,000 has been purified from Gross murine oncornavirus by phosphocellulose column chromatography. The protein was distinct from all other of the principle Gross virus components and did not react with antisera prepared against other purified Gross virus proteins with molecular weights of about 30,000, 17,000, 14,000 and 10,000. The immunologic properties of the protein were analyzed by competition radioimmunoassay. The antigenic determinants were chiefly subgroup-specific with a close homology to the analogous protein of AKR, Kirsten, or BALB/c xenotropic viruses but not to proteins of Friend, Rauscher, Moloney, NIH Swiss xenotropic, or NZB xenotropic viruses. Group-specific determinants cross-reactive with the analogous protein of these latter viruses were present but were minor components of the total antigenic specificities of the protein. Weak interspecies cross-reactivity was demonstrated with the Theilen feline virus, but very little, if any, with different primate oncornaviruses. The identity of the related, cross-reactive proteins of other murine viruses was analyzed both by competition radioimmunoassay and immunoprecipitation. It was identified as the 15,000 MW component of Rauscher and Friend viruses, and a 12,000 MW component of Moloney and BALB xenotropic viruses. The close similarity in the properties of the p15 of Rauscher murine virus and this p12 of Gross murine virus establish these as the homologous subgroup-specific proteins of the FMR and Gross subgroups of viruses.

Keratin Synthesis in SV40-Transformed Human Keratinocytes after Transient Supertransfection with Harvey Murine Sarcoma Viral DNA

A transformed cell line (HE-IS) was established by transient supertransfection of a SV40-transformed human epidermal cell culture (HE-SL) (R. T. Su and Y.-C. Chang, 1989, Exp. Cell Res. 180, 117-133) with a recombinant plasmid containing an entire Harvey murine sarcoma virus DNA. HE-IS cells showed morphological alterations and less stringent growth requirements in a defined medium. A variant line (HE-TC), selected later from the HE-IS cells after a prolonged maintenance in serum-containing Dulbecco's minimal essential medium (DMEM), was found to be able to grow in either defined or serum-containing medium. Neither HE-IS nor HE-TC cells were able to form colonies in soft agar or induce tumors in athymic mice when inoculated subcutaneously. When grown in defined medium, all transformed cells (HE-SL, HE-IS, HE-TC) revealed a similar keratin pattern as analyzed by two-dimensional gel electrophoresis. However, significant reduction of keratins (notably K5) was detected in HE-TC cells maintained in serum-containing DMEM. The keratin pattern appeared to be regulated by the growth environment. Because of their nontumorigenic characteristics and defined growth properties, these transformed keratinocytes are likely to provide a suitable model system for examining the regulation of cytoskeleton protein synthesis.