Characterization of a glycoprotein fusogen isolated from Sendai virus

After isolation from Sendai virus, the glycoproteins HN and F retained their ability to induce hemagglutination and both heterologous and homologous cell-cell fusion. Both methods for demonstrating cell fusion indicated that the isolated HN and F glycoproteins compared favorably with whole Sendai virus as a fusogen. Conditions affecting the degree of fusion were examined and optimized. Whole virus and isolated glycoprotein preparations were characterized by electron microscopy and by SDS-polyacrylamide gel electrophoresis. Lipid analysis of the glycoprotein preparations by thin layer chromatography and gas chromatography/mass spectrometry indicated that they were partially lipid-depleted during the isolation protocol and the ratio of cholesterol to phospholipid was higher than in the whole virus. A complete fatty acid analysis was performed on lipid extracts from whole virus and from glycoprotein preparations. Detergent was removed from the glycoproteins by dialysis and by incubation with Amberlite XAD-2 resin. The detergent content of the glycoprotein preparations was monitored by gas chromatography and with [3H]Triton X-100. Both methods showed that virtually all (greater than or equal to 99.8%) of the originally added detergent was removed. Electron microscopy of the negatively-stained HN and F preparations showed primarily spherical particles 120 +/- 20 A in diameter (range 80-250 A). Since no organization reminiscent of envelopes could be demonstrated, we conclude that the fusogenic activity of Sendai virus resides in the glycoproteins per se rather than in bilayer integrated lipid-protein complexes.     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of Sendai virus as fusogen

A method of introducing enzymes into the cytoplasm of fibroblasts in culture is described. Erythrocytes obtained from normal and arginase-deficient individuals were loaded with arginase in vitro and fused to arginase-deficient mouse and human fibroblasts. Erythrocyte ghost-fibroblast fusion was quantified by a 14C-radioactive assay for arginase in solubilized fibroblasts. Fusion was successfully induced by Sendai virus and also by the isolated glycoproteins of Sendai virus. After fusion the arginase activity associated with the Fibroblasts was 700--1500 U of arginase/mg of cell protein; this enzyme activity was 5- to 10-times higher than that normally found in the fibroblasts. The enrichment in arginase activity indicated that between four and ten ghosts had fused per fibroblast. The use of isolated viral proteins to mediate the transfer of enzymes into cells in vivo might alleviate clinical complications inherent in the use of whole virions. The enzyme replacement technique described in this report for a hyperargininemic model cell system should be applicable to the group of inborn errors of metabolism characterized by deficiency of an enzyme normally localized in the cytoplasmic compartment of cells.     

Sendai virus envelopes can mediate Epstein-Barr virus binding to and penetration into Epstein-Barr virus receptor-negative cells

Epstein-Barr virus (EBV) receptor-negative cells were treated with UV-inactivated Sendai virus (SV) or with reconstituted SV envelopes having a low hemolytic activity and then assayed for EBV binding or for susceptibility to EBV infection. EBV binding was assessed by using both unlabeled and fluoresceinated EBV preparations. It was found that SV or SV envelope treatment renders these cells able to bind EBV. Various experiments were performed to clarify the mechanism of this SV-induced binding. The EBV receptor-negative 1301 cells were treated with SV either at 0°C or at both 0 and 37°C successively and then examined for EBV binding at 0°C. It was thus found that when SV treatment was performed exclusively at 0°C, the target cells showed higher fluorescence intensity after their incubation with fluoresceinated EBV. In addition, Clostridium perfringens neuraminidase treatment of 1301 cells did not induce any EBV binding to these cells. These data indicate that EBV binding is not due to the disturbance of the cell membrane by SV envelope fusion or to the uncovering of EBV binding sites on the cells after the enzymatic action of SV neuraminidase. Moreover, bound EBV was partly eluted from SV-treated 1301 cells at 37°C, and the treatment of EBV with C. perfringens neuraminidase inhibited its SV-mediated binding. These data indicate that EBV binds to the hemagglutinin-neuraminidase of SV on the target cell surface and that a fraction of the bound EBV becomes irreversibly associated with the SV-treated cell membrane. Our data also show that EBV can penetrate into 1301 cells which have incorporated SV envelopes into their membrane, as demonstrated by the induction of the EBV-determined nuclear antigen by B95-8 EBV in SV envelope-treated 1301 cells.     

A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus.

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.     

Adult human RPE can be activated into a multipotent stem cell that produces mesenchymal derivatives

The retinal pigment epithelium (RPE) is a monolayer of cells underlying and supporting the neural retina. It begins as a plastic tissue, capable, in some species, of generating lens and retina, but differentiates early in development and remains normally nonproliferative throughout life. Here we show that a subpopulation of adult human RPE cells can be activated in vitro to a self-renewing cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers, proliferates extensively, and can redifferentiate into stable cobblestone RPE monolayers. Clonal studies demonstrate that RPESCs are multipotent and in defined conditions can generate both neural and mesenchymal progeny. This plasticity may explain human pathologies in which mesenchymal fates are seen in the eye, for example in proliferative vitroretinopathy (PVR) and phthisis bulbi. This study establishes the RPESC as an accessible, human CNS-derived multipotent stem cell, useful for the study of fate choice, replacement therapy, and disease modeling.     

Bacteria can be selected to help beneficial plasmids spread

A recent commentary raised concerns about aspects of the model and assumptions used in a previous study which demonstrated that selection can favor chromosomal alleles that confer higher plasmid donation rates. Here, the authors of that previous study respond to the concerns raised.

In our original work [1], we demonstrated experimentally that selection can favor chromosomal alleles that confer higher plasmid donation rates, given the plasmid is beneficial and the recipient has an elevated chance of carrying the donor allele (i.e., preferential donation to kin). Our experiments demonstrated this effect via 2 mechanisms of preferential donation: biased conjugation rates and structured populations. We interpreted these results through the lens of kin selection theory (benefits via horizontal gene transfer to kin), supported by simulations and an analytical fitness function model. These results hold importance by outlining that the evolution of plasmid transfer rates (a key aspect of the antibiotic resistance crisis) is not necessarily the sole product of selection on the plasmid itself and forms part of a broader series of papers from our labs investigating the sociomicrobiology of plasmids [2–4].A new commentary raises concerns over our fitness function model, flagging issues with both the structure of the model and assumptions made in our analysis [5]. We stand by the general conclusions of our work but accept that our fitness function and stated analysis assumptions could be better formulated. Our initial fitness function is heuristic in the sense it was designed to capture general processes acting on the fitness of individuals, dependent on the plasmid and donor allele status—without explicitly modeling the myriad demographic events of dispersal, reproduction, conjugation, and death that result in selective shifts across a metapopulation of cells. Specifically, we captured the “force of infection” faced by an uninfected cell as the product of average plasmid prevalence and average donor allele prevalence in the local patch (p_jq_j; see commentary for notation details). We agree with the authors that this force of infection is better phrased as the average of the product ((1/N)∑p_ij q_ij), in part because this avoids the potential pathology under limit conditions described by the authors, but also because this approach better highlights that the particular social trait in question is an “other only” cooperative trait [6], illustrated by commentary equation [2], where transmission to self and transmission to others are separated. This separation has the important consequence of highlighting that unlike many microbial social traits where benefits accrue to a group (including self), a cooperative plasmid donor trait can only benefit other cells that lack the plasmid. Given established costs of donation (e.g., see figure S2 in our original article), this defines our “donor” behavior as an altruistic trait, which can, therefore, only be favored by selection given nonrandom interactions among individuals (e.g., [7]).Our experimental results outline 2 mechanisms of nonrandom interactions: preferential donation to kin and population structure. Each of these mechanisms will generate positive covariances between focal individual q_ij and non-self-recipient q_j donor allele states (cov(q_j, q_ij) > 0). The pathway via preferential donation to kin (order-of-magnitude differences according to our analyses and more recent measurements among lineages coexisting within natural populations [8]) will also likely generate positive covariances between donor and recipient abilities (cov(s_ij, q_ij) > 0). In contrast, to arrive at the result that selection always works against plasmid donor alleles (equation [4]), the commentary makes the assumption that both of the above covariances are zero. We suggest that the additional analyses begun by the authors are an exciting starting point to better map selection on donor alleles, under a broader array of defined assumptions on cell–cell and gene–gene structure, ideally informed by data on structures found in natural bacterial populations.     

Hepatitis B virus can be inhibited by DNA methyltransferase 3a via specific zinc-finger-induced methylation of the X promoter

In this work we explored whether DNA methyltransferase 3a (Dnmt3a) targeted to the HBV X promoter (XP) causes epigenetic suppression of hepatitis B virus (HBV). The C-terminus of Dnmt3a (Dnmt3aC) was fused to a six-zinc-finger peptide specific to XP to form a fused DNA methyltransferase (XPDnmt3aC). The binding and methyl-modifying specificity of XPDnmt3aC were verified with an electrophoretic mobility shift assay and methylation-specific PCR, respectively. XP activity and HBV expression were clearly downregulated in HepG2 cells transfected with plasmid pXPDnmt3aC. The injection of XPDnmt3aC into HBV transgenic (TgHBV) mice also showed significant inhibition, leading to low serum HBV surface protein (HBsAg) levels and a reduced viral load. Thus, XPDnmt3aC specifically silenced HBV via site-selective DNA methylation delivered by zinc-finger peptides. This study establishes the foundation of an epigenetic way of controlling HBV-related diseases.     

The role of the target membrane structure in fusion with Sendai virus

Fusion between membranes of Sendai virus and liposomes or human erythrocytes ghosts was studied using an assay for lipid mixing based on the relief of self-quenching of octadecylrhodamine (R18) fluorescence. We considered only viral fusion that reflects the biological activity of the viral spike glycoproteins. The liposomes were made of phosphatidylcholine, and the effects of including cholesterol, the sialoglycolipid GD1a, and/or the sialoglycoprotein glycophorin as receptors were tested. Binding of Sendai virus to those liposomes at 37 degrees C was very weak. Fusion with the erythrocyte membranes occurred at a 30-fold faster rate than with the liposomes. Experiments with biological and liposomal targets of different size indicated that size did not account for differences in fusion efficiency.     

Microinjection of arginase into enzyme-deficient cells with the isolated glycoproteins of sendai virus as fusogen

Bumetanide is a potent diuretic drug which has some structural features in common with furosemide. The steady-state exchange of K⁺ and Cl^? was investigated in Ehrlich ascites tumor cells treated with bumetanide. This agent did not alter the cellular content of K⁺ or Cl^? but the self-exchange of both ions was depressed. K⁺ self-exchange was inhibited by 55% at bumetanide concentrations as low as 10^?6 M. Cl^? self-exchange was less sensitive to this drug but at low concentrations (between 10^?6 and 10^?3 M) bumetanide was a more effective inhibitor of Cl^? transfer than furosemide. The steady-state K⁺ flux of cells equilibrated in NO₃^? media was compared with the K⁺ flux in cells treated with 10^?4 or 10^?3 M bumetanide; the Cl^? -sensitive K⁺ exchange was equivalent to the bumetanide-sensitive K⁺ exchange. Since the results suggested that a bumetanide-sensitive (Cl^?, K⁺) cotransport could be operative in steady-state cells, the stoichiometry of the bumetanide-sensitive fluxes was determined by measuring Cl^? and K⁺ fluxes simultaneously in the same cell suspension. At $\text{5 · 10}{}^{\mathrm{?4}}$ and 10^?3 M bumetanide concentrations, the ratio of these fluxes was $\text{0.98 ? 0.07 (}\text{S.E.}\text{)}\text{and}\text{1.04 ? 0.06}$, respectively, consistent with the postulated cotransport mechanism. At 10^?4 and 10^?5 M, however, the ratio of the bumetanide-sensitive Cl^?/K⁺ flux was significantly less than 1.0. Since the magnitude of the bumetanide-sensitive K⁺ flux at 10^?4 M was close to that of the Cl^?-sensitive flux, a ratio of less than 1.0 at this drug level indicates that Cl^? sensitivity and drug sensitivity may not reflect inhibition of the same process under all circumstances.     

beta-catenin can be transported into the nucleus in a Ran-unassisted manner

The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion. These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner. We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.     

Stability towards alkaline conditions can be engineered into a protein ligand

One of the problems with a proteinaceous affinity ligand is their sensitivity to alkaline conditions. Here, we show that a simple and straightforward strategy consisting in replacing all asparagine residues with other amino acids can dramatically improve the chemical stability of a protein towards alkaline conditions. As a model, a Streptococcal albumin-binding domain (ABD) was used. The engineered variant showed higher stability towards 0.5 M NaOH, as well as higher thermal stability compared to its native counterpart. This protein engineering approach could potentially also be used for other protein ligands to eliminate the sensitivity to alkaline cleaning-in-place (CIP) conditions.     

The entry into host cells of Sindbis virus, vesicular stomatitis virus and Sendai virus.

We have compared the mechanisms of entry into host cells of three enveloped viruses: Sendai virus, vesicular stomatitis virus (VSV) and Sindbis virus. Virus entry by membrane fusion should antigenically modify the surface of a newly infected cell in such a way that it will be killed by anti-viral antibody and complement. On the other hand, virus entry by a mechanism involving uptake by the cell of the whole virion should not make cells sensitive to antibody and complement. As expected, cells newly infected with Sendai virus were readily and completely lysed by anti-Sendai antibody and complement. In marked contrast, however, cells newly infected with either Sindbis virus or VSV were killed by anti-viral antibody and complement only when infected at an extremely high multiplicity of infection, in excess of 1000 plaque-forming units per cell. We favor the following explanation for these results with Sindbis virus and VSV: a very large majority of the Sindbis and VSV virions entered the infected cells by some means other than membrane fusion, presumably engulfment of the whole particle. Efficient entry by way of membrane fusion may therefore not be a general characteristic of enveloped viruses.     

Sendai virus causes a rise in intracellular free Ca2+ before cell fusion

1. Sendai virus caused a large increase in the concentration of free Ca(2+) within human erythrocyte ghosts detected by the Ca(2+)-activated photoprotein obelin. 2. The increase in intracellular [Ca(2+)] preceded fusion. However, fusion could also be observed in the absence of a detectable rise in intracellular free [Ca(2+)]. 3. It was concluded that the increase in intracellular free [Ca(2+)] was not an absolute requirement for cell fusion, but may be necessary to produce fusion at the maximum rate.     

Association of ganglioside-protein conjugates into cell and Sendai virus. Requirement for the HN subunit in viral fusion

We have prepared several electron and light microscopic labels of epidermal growth factor (EGF) to analyse the morphologic features of its binding and internalization by cultured cells. These include a ferritin conjugate of EGF, a covalent conjugate of EGF and horseradish peroxidase (EGF-HRP), a colloidal gold marker system using EGF-HRP as a primary antigen, and a covalent complex of EGF with rhodamine-labelled lactalbumin. All of the light and electron microscopic labels showed similar patterns of binding. EGF initially bound to diffusely distributed cell surface receptors at 4 degrees C. The EGF-receptor complexes clustered into clathrin-coated pits on the cell surface only when the temperature was raised to 37 degrees C. In KB and Swiss 3T3 cells, this was followed by rapid internationalization into receptosomes, compartmentalization into the Golgi system, clustering in the clathrin-coated regions of the Golgi, and finally delivery into lysosomes from the Golgi. This general pathway was seen in Swiss 3T3 cells which have a low number of EGF receptors, KB cells which have a moderate number of receptors and A431 cells that have a high number of receptors. However, the ruffling activity induced in A431 cells by EGF produced some internalization through macropinosomes, making the pathway of entry more difficult to evaluate. Double label experiments showed that EGF is internalized together with alpha 2-macroglobulin and adenovirus particles. These data clarify the route of entry of EGF in different cell types using multiple labels, and shows that it enters cells through the same coated pit entry pathway as most other ligands previously examined.     

Pathways of tumour cell killing by activated macrophages: both tumour cell membrane and nucleus can be the primary target

Plasma membrane and nucleus can be primary targets of tumour cell killing by activated macrophages (AM?). Necrotic-type cytotoxicity with loss of membrane integrity and cytoplasmic swelling was expressed by AM? from normal and from perforin-deficient mice, indicating that perforin was not involved. Incubation with AM? consistently triggered the release of thymidine from prelabelled targets, whereas chromatin condensation and small DNA fragments were only occasionally detected. It is shown by means of Pulsed-Field Gel Electrophoresis that DNA degradation in target cells is a slowly progressing process that may stop at any time, indicating that nuclear-type killing doesnot necessarily lead to the formation of low molecular weight fragments. Neither Fas nor the p55 tumour necrosis factor receptor appear to be involved in signalling nuclear-type killing. Accordingly, AM? do mediate membrane- and nuclear-type killing but the mechanisms differ from those identified in T cell cytotoxicity.