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1.
Pit2 is a type III sodium-dependent phosphate transporter and the cell surface receptor for amphotropic murine leukemia virus. Indirect arguments have previously suggested that retrovirus receptor assembly play a role in triggering membrane fusion. Using CHO cells expressing physiological amounts of functional versions of human Pit2 fused to various tagging epitopes, we provide evidence that Pit2 forms assemblies at the cell surface. Living cells were exposed to cross-linking reagents and protein extracts were treated with trifluoroacetic acid (TFA), a chemical that destroys all protein interactions but covalent links. Assemblies were also detected in the absence of cross-linking and TFA treatment, indicating that they are partially resistant to detergent denaturation. The formation of homo-oligomers was documented by the coimmunoprecipitation of differently tagged molecules. The amounts of Pit2 assemblies detected in the presence or in the absence of cross-linking reagents varied with extracellular inorganic phosphate concentration ([P(i)]). Variation of signal intensity was in the range of twofold, occurred in the absence of de novo protein synthesis and took place at the cell surface. These results indicate that Pit2 assemblies exhibit variable conformations at the surface of living cells. Susceptibility to virus infection and phosphate uptake also vary with extracellular [P(i)]. A model is proposed in which cell surface Pit2 assemblies switch from a compacted to an expanded configuration in response to changes of extracellular [P(i)], and possible relationships with the variation of biological activities are discussed.  相似文献   

2.
10A1 murine leukemia virus can enter cells by using either of two different cell surface phosphate transport proteins, the gibbon ape leukemia virus receptor Glvr-1 (Pit-1) or the amphotropic retrovirus receptor Ram-1 (Pit-2). Glvr-1 and Ram-1 are widely expressed in different tissues, but the relative amounts of each are highly variable. We have developed retrovirus packaging cell lines based on 10A1 virus to take advantage of this dual receptor utilization to improve gene transfer rates in somatic cells of animals and humans, in which the relative levels of the two receptors are not always known. Optimization of the Env expression vector allowed the generation of packaging lines that produce helper-free vector titers up to 10(7)/ml. By interference analysis, we found that a 10A1 pseudotype retroviral vector can utilize Ram-1 for efficient entry into mouse, rat, and human cells and can utilize Glvr-1 for entry into mouse and human cells but not for entry into rat cells. The 10A1 pseudotype vector efficiently enters mouse cells by using Glvr-1, while entry into human cells is much less efficient. Thus, the 10A1 pseudotype packaging cells may be advantageous compared with the standard amphotropic packaging cells because vectors produced by the cells can use an additional receptor for cell entry. These packaging cells will also be useful to further explore the complicated pattern of receptor usage conferred by the 10A1 viral surface protein.  相似文献   

3.
Transduction by murine leukemia virus-based retrovirus vectors is limited in certain cell types, particularly in nondividing cells. But transduction can be inefficient even in cells that divide rapidly. For example, exposure of 208F rat embryo fibroblasts to an excess of an amphotropic retrovirus vector encoding alkaline phosphatase results in a transduction efficiency of only about 10%, even though these cells divide rapidly. Here we show that transduction of 208F cells is limited by cell surface retrovirus receptor levels; overexpression of the amphotropic retrovirus receptor Pit2 markedly improved the transduction efficiency to 50%. To characterize receptor levels and binding affinity, we synthesized a fusion protein that joins the amino terminus of the amphotropic envelope protein to the Fc region of a human immunoglobulin G1 molecule for use in binding assays. In comparison to the parental cell line, the modified cell line showed an order of magnitude increase in binding sites of from 18,000 to 150,000 per cell. Thus, efficient transduction by an amphotropic retrovirus vector requires high-level expression of the retrovirus receptor Pit2. These results provide the rationale for further examination of the role of receptor levels in inefficient transduction, especially with regard to target cells for gene therapy, where a high transduction rate is often crucial.  相似文献   

4.
The membrane receptors for the gibbon ape leukemia retrovirus and the amphotropic murine retrovirus serve normal cellular functions as sodium-dependent phosphate transporters (Pit-1 and Pit-2, respectively). Our earlier studies established that activation of protein kinase C (PKC) by treatment of cells with phorbol 12-myristate 13-acetate (PMA) enhanced sodium-dependent phosphate (Na/Pi) uptake. Studies now have been carried out to determine which type of Na/Pi transporter (Pit-1 or Pit-2) is regulated by PKC and which PKC isotypes are involved in the up-regulation of Na/Pi uptake by the Na/Pi transporter/viral receptor. It was found that the activation of short term (2-min) Na/Pi uptake by PMA is abolished when cells are infected with amphotropic murine retrovirus (binds Pit-2 receptor) but not with gibbon ape leukemia retrovirus (binds Pit-1 receptor), indicating that Pit-2 is the form of Na/Pi transporter/viral receptor regulated by PKC. The PKC-mediated activation of Pit-2 was blocked by pretreating cells with the pan-PKC inhibitor bisindolylmaleimide but not with the conventional PKC isotype inhibitor G? 6976, suggesting that a novel PKC isotype is required to regulate Pit-2. Overexpression of PKCepsilon, but not of PKCalpha, -delta, or -zeta, was found to mimic the activation of Na/Pi uptake. To further establish that PKCepsilon is involved in the regulation of Pit-2, cells were treated with PKCepsilon-selective antisense oligonucleotides. Treatment with PKCepsilon antisense oligonucleotides decreased the PMA-induced activation of Na/Pi uptake. These results indicate that PMA-induced stimulation of Na/Pi uptake by Pit-2 is specifically mediated through activation of PKCepsilon.  相似文献   

5.
Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.  相似文献   

6.
The type III sodium-dependent phosphate (NaPi) cotransporter, Pit2, is a receptor for amphotropic murine leukemia virus (A-MuLV) and 10A1 MuLV. In order to determine what is sufficient for Pit2 receptor function, a deletion mutant lacking about the middle half of the protein was made. The mutant supported entry for both viruses, unequivocally narrowing down the identification of the sequence that is sufficient to specify the receptor functions of Pit2 to its N-terminal 182 amino acids and C-terminal 170 amino acids.  相似文献   

7.
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   

8.
Mutations that negatively or positively affect the fusion properties of murine leukemia viruses (MLVs) have been found within all subdomains of their SU (surface) and TM (transmembrane) envelope units. Yet, the interrelations between these different regions of the envelope complex during the cell entry process are still elusive. Deletion of the histidine residue of the conserved PHQV motif at the amino terminus of the amphotropic or the ecotropic MLV SU resulted in the AdelH or the MOdelH fusion-defective mutant envelope, respectively. These delH mutant envelopes are incorporated on retroviral particles at normal densities and normally mediate virion binding to cells expressing the retroviral receptors. However, both their cell-cell and virus-cell fusogenicities were fully prevented at an early postbinding stage. We show here that the fusion defect of AdelH or MOdelH envelopes was also almost completely reverted by providing either soluble SU or a polypeptide encompassing the receptor-binding domain (RBD) to the target cells, provided that the integrity of the amino-terminal end of either polypeptide was preserved. Restoration of delH envelope fusogenicity was caused by activation of the target cells via specific interaction of the latter polypeptides with the retrovirus receptor rather than by their association with the delH envelope complexes. Moreover crossactivation of the target cells, leading to fusion activation of AdelH or MOdelH envelopes, was achieved by polypeptides containing various type C mammalian retrovirus RBDs, irrespective of the type of entry-defective glycoprotein that was used for infection. Our results indicate that although they recognize different receptors for binding to the cell surface, type C mammalian retroviruses use a common entry pathway which is activated by a conserved feature of their envelope glycoproteins.  相似文献   

9.
Expression of the transmembrane receptor protein Ram-1 may be critical to optimizing retroviral gene transfer. Ram-1 acts as both a sodium-dependent phosphate transporter and a receptor for amphotropic retroviruses. We previously reported detectable Ram-1 in murine hematopoietic fetal liver cells (FLC) despite resistance of these cells to amphotropic retroviral transduction (infection). We document here that Ram-1 expression is completely absent in murine yolk sac cells from days 9.5 through 13.5 of ontogeny and first appears at low levels in midgestational FLC between days 13.5 and 14.5. In addition, Ram-1 expression is detected only in more differentiated populations within FLC, day 14.5, and not in those highly enriched for stem cells, indicating developmental regulation of Ram-1 during murine hematopoiesis. Others have reported the in vitro use of phosphate-free medium as a stimulus to increase levels of Ram-1 mRNA in nonhematopoietic cells. We now demonstrate that Ram-1 poly(A)+ mRNA increases significantly following culture of FLC in phosphate-free medium. Further, transduction of FLC in phosphate-free medium with an amphotropic retrovirus containing the multiple drug resistance gene leads to gene transfer not observed previously. These data demonstrate that (i) the normal resistance of FLC to amphotropic transduction is most likely due to an insufficient number of Ram-1 molecules for efficient retroviral recognition and binding, and (ii) Ram-1 can be upregulated by increasing the need for phosphate transport across the cell membrane.  相似文献   

10.
We have engineered ecotropic Moloney murine leukemia virus-derived envelopes targeted to cell surface molecules expressed on human cells by the N-terminal insertion of polypeptides able to bind either Ram-1 phosphate transporter (the first 208 amino acids of amphotropic murine leukemia virus surface protein) or epidermal growth factor receptor (EGFR) (the 53 amino acids of EGF). Both envelopes were correctly processed and incorporated into viral particles. Virions carrying these envelopes could specifically bind the new cell surface receptors. Virions targeted to Ram-1 could infect human cells, although the efficiency was reduced compared with that of virions carrying wild-type amphotropic murine leukemia virus envelopes. The infectivity of virions targeted to EGFR was blocked at a postbinding step, and our results suggest that EGFR-bound virions were rapidly trafficked to lysosomes. These data suggest that retroviruses require specific properties of cell surface molecules to allow the release of viral cores into the correct cell compartment.  相似文献   

11.
12.
A rat cDNA (rRam-1), which was cloned on the basis that it enables Chinese hamster ovary (CHO) cells to be infected by amphotropic host range murine retroviruses, was recently found to encode a widely expressed Na(+)-phosphate symporter (M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994). CHO cells express the hamster homolog of Ram-1 but are resistant to amphotropic retroviruses. Although the amphotropic envelope glycoprotein gp70 bound weakly onto control CHO cells, CHO/rRam-1 cells had novel high-affinity binding sites, and the resulting strongly adsorbed gp70 was only slowly removed from cell surfaces, with a half-life of greater than 6 h. CHO/rRam-1 cells were also specifically and efficiently killed by exposure to amphotropic gp70 followed by antiserum to gp70 in the presence of complement. Infection with an appropriately pseudotyped form of amphotropic retrovirus 4070A did not perturb control CHO cells or inhibit their phosphate transport. In contrast, 4070A infection of CHO/rRam-1 cells caused major alterations including cell-cell fusions, a specific 40% down-modulation of the rRam-1 component of phosphate transport, and complete interference to super-infection by amphotropic viruses. The 4070A virus-infected CHO/rRam-1 cells retained a substantial cell surface pool of rRam-1 that functioned as a phosphate transporter but not as a viral receptor. We conclude that amphotropic gp70 binds more strongly to rRam-1 than to the homologous hamster protein and that this stable attachment is necessary for infection, interference, membrane fusion, and pathogenesis.  相似文献   

13.
A central feature of Salmonella pathogenicity is the bacterium's ability to enter into non-phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42- and Rac-dependent actin cytoskeleton rearrangements. These responses are triggered by the co-ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, a Salmonella inositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42-dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus, Salmonella strains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism in Salmonella-induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4 in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were not caused exclusively by SopB. We show that the SopB-independent inositol phosphate fluxes are the consequence of the SopE-dependent activation of an endogenous inositol phosphatase. The ability of Salmonella to stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.  相似文献   

14.
The amphotropic murine leukemia virus (MuLV) can infect cells from a number of mammals, including humans, via its specific receptor. Basic knowledge of amphotropic MuLV receptor expression is likely to be useful in the development and improvement of gene therapy protocols based on amphotropic-pseudotyped vectors. To investigate the expression of the human receptor for the amphotropic MuLV (GLVR-2, newly termed Pit2), we determined its mRNA levels in several cell lines and found them to vary significantly. Induction of increased levels of mRNA after removal of phosphate from the media was observed in two osteosarcoma cell lines. The increase in GLVR-2 mRNA resulted in a concomitant rise in the levels of a 71-kDa protein specifically recognized by affinity-purified antibodies against GLVR-2. Using these antibodies, we were able to confirm the intracellular topology of the large hydrophilic domain between the proposed sixth and seventh transmembrane domains of the GLVR-2 protein. This assignment is in agreement with the fourth extracellular loop being outside the cell, consistent with the proposal that the fourth extracellular loop of GLVR-2 contains the envelope binding site.  相似文献   

15.
Extracellular RNA is becoming increasingly recognized as a signaling molecule. Virally derived double stranded (ds)RNA released into the extracellular space during virus induced cell lysis acts as a powerful inducer of classical type I interferon (IFN) responses; however, the receptor that mediates this response has not been identified. Class A scavenger receptors (SR-As) are likely candidates due to their cell surface expression and ability to bind nucleic acids. In this study, we investigated a possible role for SR-As in mediating type I IFN responses induced by extracellular dsRNA in fibroblasts, a predominant producer of IFNβ. Fibroblasts were found to express functional SR-As, even SR-A species thought to be macrophage specific. SR-A specific competitive ligands significantly blocked extracellular dsRNA binding, entry and subsequent interferon stimulated gene (ISG) induction. Candidate SR-As were systematically investigated using RNAi and the most dramatic inhibition in responses was observed when all candidate SR-As were knocked down in unison. Partial inhibition of dsRNA induced antiviral responses was observed in vivo in SR-AI/II-/- mice compared with WT controls. The role of SR-As in mediating extracellular dsRNA entry and subsequent induced antiviral responses was observed in both murine and human fibroblasts. SR-As appear to function as ‘carriers’, facilitating dsRNA entry and delivery to the established dsRNA sensing receptors, specifically TLR3, RIGI and MDA-5. Identifying SR-As as gatekeepers of the cell, mediating innate antiviral responses, represents a novel function for this receptor family and provides insight into how cells recognize danger signals associated with lytic virus infections. Furthermore, the implications of a cell surface receptor capable of recognizing extracellular RNA may exceed beyond viral immunity to mediating other important innate immune functions.  相似文献   

16.
J L Battini  J M Heard    O Danos 《Journal of virology》1992,66(3):1468-1475
The envelope glycoproteins (SU) of mammalian type C retroviruses possess an amino-terminal domain of about 200 residues, which is involved in binding a cell surface receptor. In this domain, highly conserved amino acid sequences are interrupted by two segments of variable length and sequence, VRA and VRB. We have studied the role of these variable regions in receptor recognition and binding by constructing chimeric molecules in which portions of the amino-terminal domains from amphotropic (4070A), xenotropic (NZB), and polytropic (MCF 247) murine leukemia virus SU proteins were permuted. These chimeras, which exchanged either one or two variable regions, were expressed at the surface of replication-defective viral particles by a pseudotyping assay. Wild-type or recombinant env genes were transfected into a cell line producing Moloney murine leukemia virus particles devoid of envelope glycoproteins in which a retrovirus vector genome carrying an Escherichia coli lacZ gene was packaged. The host range and sensitivity to interference of pseudotyped virions were assayed, and we observed which permutations resulted in receptor switch or loss of function. Our results indicate that the determinants of receptor choice are found within the just 120 amino acids of SU proteins. Downstream sequences contribute to the stabilization of the receptor-specific structure.  相似文献   

17.
The cation-independent mannose 6-phosphate receptor (MPRCI) functions in the packaging of both newly made and extracellular lysosomal enzymes into lysosomes. The subcellular location of MPRCI reflects these two functions; receptor is found in the Golgi complex, in endosomes, and on the cell surface. To learn about the intracellular pathway followed by surface receptor and to study the relationship between the receptor pools, we examined the entry of the surface MPRCI into Golgi compartments that contain sialyltransferase. Sialic acid was removed from surface-labeled K562 cultured human erythroleukemia cells by neuraminidase treatment. When the cells were returned to culture at 37 degrees C, surface MPRCI was resialylated by the cells with a half-time of 1-2 h. Resialylation was inhibited by reduced temperature, a treatment that allows surface molecules to reach endosomes but blocks further transport. These results indicate that surface MPRCI is transported to the sialyltransferase compartment in the Golgi complex. After culture at 37 degrees C, a small fraction (10-20%) of the resialylated receptor was found on the cell surface. Because a similar fraction of the total receptor pool is found on the cell surface, it is likely that cell surface MPRCI mixes with the cellular pool after resialylation. These data also support the idea that extracellular and newly made lysosomal enzymes are transported to lysosomes through a common compartment.  相似文献   

18.
Murine retroviruses have been divided into six interference groups that use different receptors for cell entry: the ecotropic, xenotropic, polytropic, amphotropic, 10A1, and Mus dunni endogenous virus groups. Some interference is observed between xenotropic and polytropic viruses and between amphotropic and 10A1 viruses, indicating some overlap in receptor specificity between these groups, but otherwise these interference groups appear completely independent. In contrast, one study found interference among many of these groups when Mus dunni wild mouse cells were examined with an immunofluorescence assay to detect infection by the challenge virus. Here we have used a more direct assay for cell entry by using pseudotyped retroviral vectors to measure interference in M. dunni cells, and we find no evidence for extensive interference between members of different murine retrovirus groups. Indeed, our results in M. dunni cells are consistent with interference results observed in other cell types and indicate that the anomalous interference results previously observed in M. dunni cells with the immunofluorescence assay were most likely due to factors other than those that affect receptor-mediated virus entry. In summary, our results show that murine retroviruses use at least six different receptors for entry into M. dunni cells.  相似文献   

19.
Retrovirus entry into cells is mediated by specific interactions between the retrovirally encoded Env envelope glycoprotein and a host cell surface receptor. Though a number of peptide motifs responsible for the structure as well as for the binding and fusion activities of Env have been identified, only a few quantitative data concerning the infection process are available. Using an inducible expression system, we have expressed various amounts of ecotropic and amphotropic Env at the surfaces of Moloney murine leukemia virus-derived vectors and assayed for the infectivity of viral particles. Contrary to the current view that numerous noncooperative Env-viral receptor interactions are required for cell infection, we report here that very small amounts of Env are sufficient for optimal infection. However, increasing Env density clearly accelerates the rate at which infectious attachment to cells occurs. Moreover, our data also show that a surprisingly small number of Env molecules are sufficient to drive infection, albeit at a reduced efficiency, and that, under conditions of low expression, Env molecules act cooperatively. These observations have important consequences for our understanding of natural retroviral infection as well as for the design of cell-targeted infection techniques involving retroviral vectors.  相似文献   

20.
We describe the development of an experimental system to test the hypothesis that the efficiency of retrovirus transduction is dependent on the pathway of virus entry into the host cell and the intracellular trafficking itinerary of the cellular receptor with which it interacts. The experimental system consists of three model target cell lines, derived from HeLa cells, that stably express one of three interleukin-2 receptor alpha chain (CD25) chimeras, TAC, TAC-CD16, and TAC-DKQTLL, which have identical extracellular domains but different intracellular trafficking itineraries, and a targeted amphotropic murine leukemia retrovirus whose envelope proteins were modified to include a binding site for TAC at their N-termini. We found that the efficiency of retrovirus transduction was affected by the distribution and trafficking itinerary of the TAC receptors. Transduction of cells that expressed TAC-DKQTLL was nearly 4-fold lower than transduction of control cells that did not express any of the TAC receptors. In contrast, transduction of cells that expressed TAC was 1.6-fold higher than transduction of control cells, whereas transduction was not significantly affected by the expression of TAC-CD16. Our results suggest that in the course of designing a targeted retrovirus it may be prudent to target only those receptors that internalize retroviruses via pathways that most efficiently support post-binding steps of infection.  相似文献   

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