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1.
Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes. 总被引:5,自引:4,他引:1
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The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup. 相似文献
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Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells. 总被引:1,自引:0,他引:1
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K Qing T Bachelot P Mukherjee X S Wang L Peng M C Yoder P Leboulch A Srivastava 《Journal of virology》1997,71(7):5663-5667
The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy. 相似文献
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Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors. 总被引:6,自引:6,他引:0
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J Bertran J L Miller Y Yang A Fenimore-Justman F Rueda E F Vanin A W Nienhuis 《Journal of virology》1996,70(10):6759-6766
Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration. 相似文献
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Proto-oncogene expression in regenerating liver is simulated in cultures of primary adult rat hepatocytes 总被引:20,自引:0,他引:20
W Kruijer H Skelly F Botteri H van der Putten J R Barber I M Verma H L Leffert 《The Journal of biological chemistry》1986,261(17):7929-7933
Proto-oncogene fos mRNA levels are rapidly and transiently elevated 12-fold in regenerating liver 10-60 min following partial hepatectomy. This response, and the induction of fos protein synthesis, has been simulated qualitatively and quantitatively in long term primary cultures of quiescent adult rat hepatocytes where proliferative transitions can be initiated directly in serum-free medium by known hepatocyte mitogens like epidermal growth factor. Expression of a second proto-oncogene, c-rasH, in proliferatively activated hepatocyte cultures between 6 and 24 h also simulates the delayed hepatic response that occurs in vivo following partial hepatectomy. These results suggest that sequential proto-oncogene expression during liver regeneration is caused directly by hepatocellular interactions with specific mitogens. In addition, a role for monovalent cations in the regulation of hepatocyte gene expression is implicated from findings that Na+ deprivation inhibits induction of fos expression in cultured hepatocytes by epidermal growth factor under chemically defined conditions. 相似文献
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Increase of transferrin receptors in regenerating rat liver cells after partial hepatectomy 总被引:1,自引:0,他引:1
I Tei Y Makino T Kadofuku I Kanamaru K Konno 《Biochemical and biophysical research communications》1984,121(2):717-721
The change of transferrin receptors in regenerating rat liver cells after partial hepatectomy was demonstrated. The binding of 125I-labeled transferrin to the cells began to increase after 24 h of partial hepatectomy and reached about double that of the non-operated normal rat liver cells. A Scatchard analysis of binding parameters showed 1.62 X 10(5) (Ka: 1.04 X 10(7) M-1) in normal cells and 3.36 X 10(5) (Ka: 1.23 X 10(7) M-1) in regenerating cells. This reflected on increase of transferrin receptor number and little change occurred in the binding affinity between transferrin and its surface receptor. 相似文献
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The use of histone as a facilitator to improve the efficiency of retroviral gene transfer.
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Vectors based on murine C-type retroviruses are commonly used in biology. The efficiency of viral infection is normally increased by a facilitator, for example polybrene, DEAE-dextran or a liposome. The receptor for ecotropic viruses is a transporter for basic amino acids; we therefore explored the use of a highly basic protein, histone type IIA, as a facilitator. We show in several cell types that histone is as efficient as the other agents tested, and in some cases more so. This readily available reagent is thus likely to be useful in the wide range of studies that employ retroviral vectors. 相似文献
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Chen Z Kolokoltsov AA Wang J Adhikary S Lorinczi M Elferink LA Davey RA 《Journal of virology》2012,86(3):1421-1432
For retroviruses such as HIV-1 and murine leukemia virus (MLV), active receptor recruitment and trafficking occur during viral entry. However, the underlying mechanisms and cellular factors involved in the process are largely uncharacterized. The viral receptor for ecotropic MLV (eMLV), a classical model for retrovirus infection mechanisms and pathogenesis, is mouse cationic amino acid transporter 1 (mCAT-1). Growth factor receptor-bound protein 2 (GRB2) is an adaptor protein that has been shown to couple cell surface receptors, such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor, to intracellular signaling events. Here we examined if GRB2 could also play a role in controlling infection by retroviruses by affecting receptor function. The GRB2 RNA interference (RNAi)-mediated suppression of endogenous GRB2 resulted in a consistent and significant reduction of virus binding and membrane fusion. The binding between eMLV and cells promoted increased GRB2-mCAT-1 interactions, as detected by immunoprecipitation. Consistently, the increased colocalization of GRB2 and mCAT-1 signals was detected by confocal microscopy. This association was time dependent and paralleled the kinetics of cell-virus membrane fusion. Interestingly, unlike the canonical binding pattern seen for GRB2 and growth factor receptors, GRB2-mCAT-1 binding does not depend on the GRB2-SH2 domain-mediated recognition of tyrosine phosphorylation on the receptor. The inhibition of endogenous GRB2 led to a reduction in surface levels of mCAT-1, which was detected by immunoprecipitation and by a direct binding assay using a recombinant MLV envelope protein receptor binding domain (RBD). Consistent with this observation, the expression of a dominant negative GRB2 mutant (R86K) resulted in the sequestration of mCAT-1 from the cell surface into intracellular vesicles. Taken together, these findings suggest a novel role for GRB2 in ecotropic MLV entry and infection by facilitating mCAT-1 trafficking. 相似文献
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Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses. 总被引:35,自引:29,他引:6
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Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses. 相似文献
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Exceptional fusogenicity of Chinese hamster ovary cells with murine retroviruses suggests roles for cellular factor(s) and receptor clusters in the membrane fusion process. 总被引:6,自引:3,他引:3
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Chinese hamster ovary (CHO) cells are naturally resistant to infection by amphotropic and ecotropic murine retroviruses, but they become susceptible after expressing corresponding receptors rRAM-1 and mCAT-1, respectively, and they then form abundant syncytia when exposed to these viruses. The fusogenic activities of CHO cell clones increase much more strongly with levels of receptor expression than do their susceptibilities to infection, suggesting that the assembly of receptor clusters may limit syncytium formation. However, other cell lines are not fusogenic, even if they express larger amounts of receptors. Our results suggest that a factor that is relatively abundant or active in CHO cells may functionally interact with rRAM-1 and mCAT-1 in a pathway that enables receptor-bearing membranes to fuse with membranes that contain viral envelope glycoproteins. In the case of CHO/rRAM-1 cells, syncytia form at foci of amphotropic 4070A virus infection by fusion-from-within of infected with uninfected cells. This fusogenic propensity is a sole property of the uninfected CHO/rRAM-1 cells, which fuse in cocultures with any cells infected with 4070A virus. With CHO/mCAT-1 cells, fusogenicity is even greater and involves fusion-from-without by ecotropic virion particles. In contrast to infection, which behaves as expected for a process limited by ecotropic virus attachment to single receptors, fusion-from-without increases dramatically for cells that express the highest levels of mCAT-1. We propose that infection and syncytium formation are limited at distinct steps of a common pathway that requires virus binding to a single receptor, assembly of multivalent virus-receptor complexes, structural changes in viral envelope glycoproteins, and membrane fusion. The limiting step in syncytium formation is a cellular process that depends on receptor clustering and is relatively active in CHO cells. 相似文献
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Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis. 总被引:3,自引:0,他引:3
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Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. 相似文献
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Ursula Glück JosLuis Rodríguez Fernndez Roumen Pankov Avri Ben-Ze'ev 《Experimental cell research》1992,202(2):477-486
The expression of the adherens junction proteins vinculin, alpha-actinin, and talin was compared in serum-stimulated 3T3 cells and in regenerating rat liver following partial hepatectomy. The levels of vinculin RNA and protein synthesis were rapidly and transiently elevated in growth-activated fibroblasts (peaking at 2-3 h) and in regenerating liver (at 4-8 h), preceding the replicative stage. alpha-Actinin expression was also induced, but more slowly (peaking at 6-8 h in 3T3 cells and at 28 h in regenerating liver), and remained elevated when DNA synthesis was proceeding in both systems. The expression of talin RNA was only slightly elevated in 3T3 cells following serum stimulation, and it remained largely unchanged in regenerating liver. The levels of RNA coding for fibronectin and for the beta 1-integrin subunit were transiently and extensively induced during liver regeneration (fibronectin with a peak at 8 h and beta 1-integrin at 12 h). The uvomorulin RNA level, and the expression of the liver-specific genes albumin and transthyretin, decreased in regenerating liver. The results suggest a physiologically significant regulation in the expression of structural components which link the extracellular matrix to the microfilament system in growth-activated fibroblasts and in regenerating liver. 相似文献
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Zweifel M Breu K Matozan K Renner E Welle M Schaffner T Clavien PA 《Immunology and cell biology》2005,83(6):587-595
Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass. 相似文献
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