5 results on '"Burimski I"'
Search Results
2. Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation.
- Author
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Yunus AS, Jackson TP, Crisafi K, Burimski I, Kilgore NR, Zoumplis D, Allaway GP, Wild CT, and Salzwedel K
- Subjects
- Circular Dichroism, Enzyme-Linked Immunosorbent Assay, Hot Temperature, Humans, In Vitro Techniques, Protein Structure, Secondary, Recombinant Proteins metabolism, Respiratory Syncytial Virus Infections metabolism, Respiratory Syncytial Virus, Human metabolism, Viral Fusion Proteins chemistry
- Abstract
Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.
- Published
- 2010
- Full Text
- View/download PDF
3. Breeding of retroviruses by DNA shuffling for improved stability and processing yields.
- Author
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Powell SK, Kaloss MA, Pinkstaff A, McKee R, Burimski I, Pensiero M, Otto E, Stemmer WP, and Soong NW
- Subjects
- Animals, Cell Line, Mice, Moloney murine leukemia virus genetics, Ultracentrifugation, Virus Replication, Directed Molecular Evolution methods, Gene Products, env genetics, Genetic Vectors, Moloney murine leukemia virus isolation & purification, Moloney murine leukemia virus physiology, Recombination, Genetic
- Abstract
Manufacturing of retroviral vectors for gene therapy is complicated by the sensitivity of these viruses to stress forces during purification and concentration. To isolate viruses that are resistant to these manufacturing processes, we performed breeding of six ecotropic murine leukemia virus (MLV) strains by DNA shuffling. The envelope regions were shuffled to generate a recombinant library of 5 x 106 replication-competent retroviruses. This library was subjected to the concentration process three consecutive times, with amplification of the surviving viruses after each cycle. Several viral clones with greatly improved stabilities were isolated, with the best clone exhibiting no loss in titer under conditions that reduced the titers of the parental viruses by 30- to 100-fold. The envelopes of these resistant viruses differed in DNA and protein sequence, and all were complex chimeras derived from multiple parents. These studies demonstrate the utility of DNA shuffling in breeding viral strains with improved characteristics for gene therapy.
- Published
- 2000
- Full Text
- View/download PDF
4. In vitro analysis of transformation potential associated with retroviral vector insertions.
- Author
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Powell SK, Kaloss M, Burimski I, Weaver L, Long Z, Lyons R, McGarrity GJ, and Otto E
- Subjects
- 3T3 Cells, Animals, Cell Line, Transformed, Flow Cytometry, Lymphocytes virology, Mice, Cell Transformation, Viral genetics, Genetic Vectors, Retroviridae genetics
- Abstract
While replication-defective retroviral vectors provide excellent vehicles for the long-term expression of therapeutic genes, they also harbor the potential to induce undesired genetic changes by random insertions into the host genome. The rate of insertional mutagenesis for retroviral vectors has been determined in several different assay systems; however, the rate at which such events induce cellular transformation has not been directly determined. Such measurements are critical to determining the actual risk of carcinogenesis resulting from retroviral gene therapy. In this study, the ability of a replication-defective retroviral vector, GlnBgSvNa, to induce cellular transformation in the BALB/c-3T3 in vitro transformation assay was assessed. The transformation frequency observed in vector-transduced BALB/c-3T3 cells, which contained one to six copies of integrated provirus, was not significantly different from that of untreated control cells. The finding that GlnBgSvNa was nontransforming in this assay indicates that the rate of transformation induced by retroviral insertions is less than the spontaneous rate of cellular transformation by BALB/c-3T3 cells, or less than 1.1 x 10(-5). These results are the first to define an upper limit for the rate of transformation induced by retroviral vectors.
- Published
- 1999
- Full Text
- View/download PDF
5. Biosafety monitoring of patients receiving intracerebral injections of murine retroviral vector producer cells.
- Author
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Long Z, Li LP, Grooms T, Lockey C, Nader K, Mychkovsky I, Mueller S, Burimski I, Ryan P, Kikuchi G, Ennist D, Marcus S, Otto E, and McGarrity G
- Subjects
- Animals, Antibodies, Viral biosynthesis, Clinical Trials as Topic, Humans, Mice, Microinjections, Multicenter Studies as Topic, Polymerase Chain Reaction, United States, United States Food and Drug Administration, Brain Neoplasms therapy, Genetic Therapy adverse effects, Genetic Vectors, Monitoring, Physiologic methods, Retroviridae genetics, Virus Replication
- Abstract
Patients with recurrent malignant brain cancer, who were receiving gene therapy by intracerebral injection of murine retroviral vector producer cells (VPCs), were monitored for the presence of replication-competent retrovirus (RCR). RCR sequences were not detected by polymerase chain reaction (PCR) in any of the 608 peripheral blood leukocyte (PBL) samples analyzed. Vector DNA sequences were detected transiently in PBL samples from a subset of 34 patients. Humoral immune responses to a retroviral core protein p30 and murine VPC were detected in some patients, most frequently in patients receiving repeated administrations of VPC. RCR was not detected in biological assays of PBLs from 41 patients who had either anti-retroviral antibodies in sera and/or vector DNA in PBLs. Our data suggest that in situ generation of RCR was not detected following intracerebral inoculation of VPCs in any of the 128 patients evaluated.
- Published
- 1998
- Full Text
- View/download PDF
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