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S J Russell

Publications and source records attributed to S J Russell.

At least 55 records · Page 3Linked to original sources
PubMed2000

A lentiviral vector expressing a fusogenic glycoprotein for cancer gene therapy.

The gibbon ape leukaemia virus envelope fusogenic membrane glycoprotein (GALV FMG) is a highly potent cytotoxic gene with great potential for use in cancer gene therapy. Here, we show that production of a VSV-G pseudotyped lentiviral vector expressing GALV FMG reconciles the requirements of viral production with the cytotoxic effects of GALV in human cells and has high titres on both dividing and quiescent tumour cells. Direct intratumoral injection of these stocks eradicated progressively growing human tumour xenografts. The potent bystander effect of the FMG transgene is a major contributor to the success of this approach but immunological activation may also be a factor. To our knowledge, this is the first demonstration in vivo of the potential both of FMG and lentiviral vectors for cancer gene therapy and highlights the importance of exploring different vector systems to complement the biological properties of the therapeutic transgene.

Animals↗
PubMed2000

Activation of a cell entry pathway common to type C mammalian retroviruses by soluble envelope fragments.

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.

Animals↗
PubMed2000

Recombinant measles viruses efficiently entering cells through targeted receptors.

We sought proof of principle that one of the safest human vaccines, measles virus Edmonston B (MV-Edm), can be genetically modified to allow entry via cell surface molecules other than its receptor CD46. Hybrid proteins consisting of the epidermal growth factor (EGF) or the insulin-like growth factor 1 (IGF1) linked to the extracellular (carboxyl) terminus of the MV-Edm attachment protein hemagglutinin (H) were produced. The standard H protein gene was replaced by one coding for H/EGF or H/IGF1 in cDNA copies of the MV genome. Recombinant viruses were rescued and replicated to titers approaching those of the parental strain. MV displaying EGF or IGF1 efficiently entered CD46-negative rodent cells expressing the human EGF or the IGF1 receptor, respectively, and the EGF virus caused extensive syncytium formation and cell death. Taking advantage of a factor Xa protease recognition site engineered in the hybrid H proteins, the displayed domain was cleaved off from virus particles, and specific entry in rodent cells was abrogated. These studies prove that MV can be engineered to selectively eliminate cells expressing a targeted receptor and provide insights into the mechanism of MV entry.

3T3 Cells↗
PubMed1999

Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.

Amino Acid Sequence↗
PubMed1999

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.

Cell Cycle↗
PubMed1999

Retroviral display of functional binding domains fused to the amino terminus of influenza hemagglutinin.

We have previously shown that retroviral vector particles derived from Moloney murine leukemia virus (Mo-MuLV) can efficiently incorporate influenza hemagglutinin (HA) glycoproteins from fowl plague virus (FPV), thus conferring a broad tropism to the vectors. To modify its host range, we have engineered the FPV HA to display four different polypeptides on its N terminus: the epidermal growth factor, an anti-human MHC class I molecules scFv (single-chain antibody), an anti-melanoma antigen scFv, and an IgG Fc-binding polypeptide. All recombinant HA glycoproteins were correctly expressed and processed, and efficiently incorporated into Mo-MuLV retroviral particles, indicating that amino-terminal insertion of large polypeptides did not alter the conformation of HA chimeras. Virions carrying the different chimeras bound specifically to cells expressing the targeted cell surface molecules of each ligand. In addition, all virion types were infectious but exhibited various degrees of specificity regarding the use of the targeted cell surface molecule versus the wild-type FPV HA receptor for cell entry and infection. For some ligands tested, infectivity was significantly increased on cells that express the targeted receptor, compared with cells that express only the wild-type HA receptor. Furthermore, some polypeptides could abolish infectivity via the wild-type FPV HA receptor. Our data therefore indicate that it is possible to engineer the HA envelope glycoprotein by fusing ligands to its amino-terminal end without affecting its fusion activity.

3T3 Cells↗
PubMed1999

Efficient discrimination between different densities of target antigen by tetracycline-regulatable T bodies.

Engineered T cells expressing chimeric T cell receptors (chTCRs) are of interest for cancer gene therapy but many "cancer antigens" are thought to be unsuitable targets because they are expressed at low levels on normal tissues. We therefore sought to determine whether engineered T cells expressing variable surface densities of a high-affinity chTCR could discriminate different concentrations of the targeted antigen. We plotted the relationship between chTCR density and the concentration of target antigen using Jurkat T cells expressing a hapten-binding chTCR whose expression could be modulated by tetracycline. Our analysis reveals that there is a dynamic equilibrium between cell surface density of the chTCR and the antigen density that optimally triggers T cell activation. At a fixed density of target antigen, optimal T cell activation can be achieved only within a certain range of chTCR densities, while excessive TCR signaling triggers apoptosis of the engineered T cells. Our results show that T cells can be engineered to discriminate different antigen densities and that the T cell response to a fixed concentration of antigen can be optimized by tuning the cell surface density of TCRs.

Antigens, Neoplasm↗
PubMed1999

Modification of retroviral tropism by display of IGF-I.

We have displayed insulin-like growth factor I (IGF-I) as an N-terminal extension of 4070A (amphotropic) retroviral envelope protein. Western blot demonstrated that chimaeric envelope proteins were incorporated into retroviral particles. Interaction between the displayed IGF-I and cell-surface receptors impaired gene delivery. The magnitude of this inhibitory effect was smallest on NIH 3T3 cells, greater on NIH 3T3 cells over-expressing insulin receptor, and greatest on NIH 3T3 cells over-expressing human type-I IGF receptor. Hence, both the number of ligand receptors and their affinity for the displayed ligand influenced the level of gene delivery. The inhibitory effect was abrogated by cleaving the displayed domain from the underlying envelope protein with factor Xa protease, and by the addition of free ligand to the infection. Addition of IGF-I or insulin caused a dose-dependent increase in titre. Possible mechanisms for receptor-mediated inhibition of gene delivery by IGF-displaying vectors are discussed, together with the implications of these results for practical applications of retroviral display and for understanding the mechanism of virus entry.

3T3 Cells↗
PubMed1999

Complement depletion does not reduce brain injury in a rabbit model of thromboembolic stroke.

The contribution of the complement system to cerebral ischemic and ischemia/reperfusion injury was examined in a rabbit model of thromboembolic stroke by delivery of an autologous clot embolus to the intracranial circulation via the internal carotid artery. A two-by-two factorial design was employed to study the impact of complement depletion via pretreatment with cobra venom factor (CVF, 100 U/kg i.v.) in the setting of permanent (without tissue plasminogen activator; t-PA) and transient (with t-PA) cerebral ischemia. Thirty-two New Zealand white rabbits were assigned to one of four groups (n=8, each group): control without t-PA, control with t-PA, CVF without t-PA and CVF with t-PA. In the complement intact animals, t-PA administration resulted in an approximate 30% reduction in infarct size when compared to the group not receiving t-PA (20.4+/-6.6% of hemisphere area vs. 30.1+/-7.2%; mean+/-SEM). However, infarct sizes in the complement depleted rabbits, with (30.7+/-8.2%) or without (30.2+/-7.9%) t-PA, were no different from the control group receiving no therapy. Similarly, no difference in regional cerebral blood flow or final intracranial pressure values was noted between any of the four groups. Complement activation does not appear to be a primary contributor to brain injury in acute thromboembolic stroke.

Animals↗
PubMed1999

Viral vector targeting.

The field of viral vector targeting is advancing rapidly. Recent advances include the successful use of bifunctional crosslinkers to target adenoviral and retroviral vectors, elucidation of the crystal structures of an adenoviral and a retroviral receptor-binding domain, and definition of strategies for inserting short targeting peptides and larger polypeptide-binding domains into the coat proteins of a number of different viral vectors. Novel targeting strategies based on host range restriction and protease activation have been developed, targeted replication-competent vectors have shown promise as anti-cancer agents and the possibility of selecting targeted vectors from vector display libraries has been established.

Animals↗
PubMed1999

The 19S regulatory complex of the proteasome functions independently of proteolysis in nucleotide excision repair.

The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes. The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae. Deletion of the ubiquitin-like domain causes UV radiation sensitivity. Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system. Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo. Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER. This establishes that the regulatory complex of the proteasome has a function independent of protein degradation.

Acetylcysteine↗
PubMed1998

Inverse targeting of retroviral vectors: selective gene transfer in a mixed population of hematopoietic and nonhematopoietic cells.

We previously reported that retroviral vectors displaying epidermal growth factor (EGF) as part of a chimeric envelope glycoprotein are sequestered upon binding to EGF receptor (EGFR)-positive target cells, leading to loss of infectivity. In the current study, we have displayed stem cell factor (SCF) on beta-galactosidase-transducing ecotropic and amphotropic retroviral vector particles as a factor Xa protease-cleavable N-terminal extension of the envelope glycoprotein. Viral incorporation of the SCF chimeric envelopes was demonstrated by immunoblotting of pelleted virions and their specific attachment to Kit receptors was demonstrated by flow cytometry. Gene transfer studies showed that when SCF was displayed on an amphotropic envelope, the infectivity of the SCF-displaying vectors was selectively inhibited on Kit-expressing cells, but could be restored by adding soluble SCF to block the Kit receptors or by cleaving the displayed SCF domain from the vector particles with factor Xa protease. The host range properties of EGF-displaying and SCF-displaying vectors were then compared in cell mixing experiments. When EGFR-positive cancer cells and Kit-positive hematopoietic cells were mixed and exposed to the different engineered vector particles, the cancer cells were selectively transduced by the SCF-displaying vector and the hematopoietic cells were selectively transduced by the EGF-displaying vector. Retroviral display of polypeptide growth factors can therefore provide the basis for a novel inverse targeting strategy with potential use for selective transduction of hematopoietic or nonhematopoietic cells (eg, cancer cells) in a mixed cell population.

3T3 Cells↗
PubMed1998

In vivo selection of protease cleavage sites from retrovirus display libraries.

Phage display libraries are widely used for selection and optimization of polypeptide ligands or protease substrates. Because they are expressed and amplified in bacterial hosts, phage are not ideal for displaying eukaryotic polypeptides or for probing mammalian cells. As retroviruses do not suffer from these limitations we constructed plasmids encoding replication-competent murine leukemia viruses displaying a virally encoded epidermal growth factor (EGF) domain at the N-terminus of the envelope glycoprotein. The EGF-displaying viruses replicated freely on EGF receptor-poor cells without deleting the displayed EGF domain but did not propagate on EGF receptor-rich cells because they were sequestered by the EGF receptors. A retrovirus display library was then generated by diversifying the seven-residue linker between the envelope glycoprotein and the displayed EGF domain. Selective pressure for loss of EGF receptor-binding activity was applied to the library by serial passage on EGF receptor-rich HT1080 cells. The selected viruses propagated on these cells with wild-type efficiencies, a phenotype that was conferred by intracellular cleavage of their displayed linker sequences. The selected linker sequences invariably presented arginine-rich motifs matching the consensus cleavage signal for furin-like proteases. Retrovirus display libraries can be used for the selection of polypeptides interacting with components of living mammalian cells.

3T3 Cells↗
PubMed1998

A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes.

The entry of retroviruses into cells depends on receptor recognition by the viral envelope surface subunit SU followed by membrane fusion, which is thought to be mediated by a fusion peptide located at the amino terminus of the envelope transmembrane subunit TM. Several fusion determinants have been previously identified in murine leukemia virus (MLV) envelopes, but their functional interrelationships as well as the processes involved in fusion activation upon retroviral receptor recognition remain unelucidated. Despite both structural and functional similarities of their envelope glycoproteins, ecotropic and amphotropic MLVs display two different postbinding properties: (i) while amphotropic MLVs fuse the cells at neutral pH, penetration of ecotropic MLVs is relatively acid pH dependent and (ii) ecotropic envelopes are more efficient than amphotropic envelopes in inducing cell-to-cell fusion and syncytium formation. By exploiting the latter characteristic in the analysis of chimeras of ecotropic and amphotropic MLV envelopes, we show here that substitution of the ecotropic MLV proline-rich region (PRR), located in the SU between the amino-terminal receptor binding domain and the TM-interacting SU carboxy-terminal domains, is sufficient to revert the amphotropic low-fusogenic phenotype into a high-fusogenic one. Furthermore, we have identified potential beta-turns in the PRR that control the stability of SU-TM associations as well as the thresholds required to trigger either cell-to-cell or virus-to-cell fusion. These data, demonstrating that the PRR functions as a signal which induces envelope conformational changes leading to fusion, have enabled us to derive envelopes which can infect cells harboring low levels of available amphotropic receptors.

Amino Acid Sequence↗
PubMed1998

Incorporation of fowl plague virus hemagglutinin into murine leukemia virus particles and analysis of the infectivity of the pseudotyped retroviruses.

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.

Animals↗
PubMed1997

Pharmacological control of antigen responsiveness in genetically modified T lymphocytes.

A chimeric TCR gene, comprising an anti-hapten single-chain Ab variable fragment fused to the transmembrane and cytoplasmic regions of the human TCR zeta-chain, was used to determine whether the tetracycline-regulatable system could be used to regulate gene expression in T cells. Jurkat T cells were stably transfected with a single vector encoding the tetracycline trans-activator protein, controlled by a constitutive promoter, and the chimeric TCR, under the control of a trans-activator protein-responsive promoter. In the absence of tetracyclines, the transfected T cells were shown to express the chimeric receptor on the cell surface and could be activated by its cognate Ag, leading to the secretion of IL-2. When the cells were exposed to increasing concentrations of tetracyclines, surface expression of the chimeric receptor was suppressed in a dose-dependent manner, and this suppression was sufficient to result in complete loss of responsiveness to the targeted Ag. Prolonged suppression of trans-gene expression for up to 7 days was observed after doxycycline was removed from the cultures, but eventual recovery of surface expression was complete, and the absolute time to recovery was directly proportional to the initial concentration of the drug. Pharmacologic control of trans-gene expression in gene-modified T cells will not only facilitate new approaches to the study of different aspects of T cell biology, but will also provide the basis for new gene therapy strategies.

Antigens↗
PubMed1997

Masking of retroviral envelope functions by oligomerizing polypeptide adaptors.

We have constructed chimeric retroviral envelopes displaying N-terminal polypeptides that are known to form homotrimeric associations. The amphotropic receptor (RAM-1) binding domain from the trimeric surface (SU) glycoprotein of 4070A murine leukemia virus (MLV)-inhibited ecotropic receptor (Rec-1) mediated infection by the SU glycoprotein of Moloney MLV when grafted to its N-terminus. The block to Rec-1-mediated infection was reversed when the RAM-1 binding domain was cleaved from the vector particles using an engineered factor Xa protease-sensitive cleavage signal between the envelope glycoprotein and its N-terminal extension. Trimeric leucine zipper peptides and the trimeric C-terminal domain of CD40 ligand were shown to inhibit RAM-1-mediated infection of NIH3T3 cells by the 4070A envelope when fused to its N-terminus, whereas monomeric helical peptides and the monomeric epidermal growth factor domain did not. The block to RAM-1-mediated infection was reversed when the trimeric polypeptides were cleaved from the vector particles by addition of factor Xa protease. Envelope binding assays using cleaved and uncleaved chimeric 4070A envelopes revealed that binding to RAM-1 receptors on mammalian cells was hindered by trimeric, but not by monomeric, N-terminal polypeptides. These results have important implications for the design of protease-activatable vectors for targeted gene delivery.

3T3 Cells↗