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osu1307730650.pdf (3.48 MB)
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Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State, a Tool to Study Protein Triggering
Author Info
Chaiwatpongsakorn, Supranee
Permalink:
http://rave.ohiolink.edu/etdc/view?acc_num=osu1307730650
Abstract Details
Year and Degree
2011, Doctor of Philosophy, Ohio State University, Comparative and Veterinary Medicine.
Abstract
Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, Pneumovirinae subfamily is the most significant respiratory pathogen in infants and second only to influenza virus in the elderly. Despite extensive efforts, no vaccines or small molecule antiviral drugs are available. The RSV fusion (F) glycoprotein has been a major target for vaccine and antiviral drug development because of its importance in the viral replication cycle, its conserved sequence and structure, its exposed position in the virion, and its strong immunogenicity. Like other paramyxoviruses, the RSV F protein is anchored in the virion membrane in a metastable, pretriggered form. Once triggered, the F protein undergoes a dramatic conformational extension that inserts its hydrophobic fusion peptide into the target cell membrane, then folds back on itself to bring the membranes together and initiate fusion. However, the Pneumovirinae F protein is unique in that it, alone, is sufficient to mediate membrane fusion and virus infection. It is, therefore, the simplest F protein to study. It likely has the ability to attach to target cells from which position it is triggered. Neither the trigger site on the F protein nor the triggering molecule/event has been identified. To begin to study the triggering mechanism of the RSV F protein biochemically, we have generated a soluble F (sF) protein by replacing the transmembrane and cytoplasmic tail domains with a 6His tag. This sF protein is secreted efficiently from 293T cells in a fully cleaved form. It is recognized by neutralizing monoclonal antibodies, appears spherical by electron microscopy, and is not aggregated, all consistent with a native, pretriggered trimer. The sF protein was purified on a Ni2+ column and eluted with 50 mM phosphate buffer containing 500 mM NaCl and 250 mM imidazole. Dialysis against 10 mM buffer caused the sF protein to trigger, forming “hatpin” shaped molecules that aggregated as rosettes, characteristic of the posttriggered form. Further dialysis experiments indicated that the efficiency of triggering correlated well with the reduction of buffer molarity. Reduction of buffer molarity by dilution also resulted in exposure of the fusion peptide as detected by liposome association, confirming sF protein triggering. Mutation of the furin cleavage site adjacent to the fusion peptide prevented liposome association, confirming that association is via the fusion peptide. Although it is not clear whether reduction in molarity can serve as a physiological trigger of the intact F protein during the natural infection of RSV, our study has revealed a novel, surrogate method for triggering a viral fusion protein. The availability of pretriggered RSV sF protein capable of being triggered and transformation into its posttriggered conformation enables studies of its mechanism of attachment, triggering, and refolding, a protein vaccine for adults, assays to quantify antibodies against F, discovery of the mechanism of action of drugs known to target F, and high throughput screens to identify new and better drugs against F.
Committee
Mark Peeples, PhD (Advisor)
Michael Oglesbee, PhD (Committee Member)
Stefan Niewiesk, PhD (Committee Member)
Jianrong Li, PhD (Committee Member)
Pages
160 p.
Subject Headings
Virology
Keywords
RSV
;
fusion protein
;
triggering
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Citations
Chaiwatpongsakorn, S. (2011).
Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State, a Tool to Study Protein Triggering
[Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1307730650
APA Style (7th edition)
Chaiwatpongsakorn, Supranee.
Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State, a Tool to Study Protein Triggering.
2011. Ohio State University, Doctoral dissertation.
OhioLINK Electronic Theses and Dissertations Center
, http://rave.ohiolink.edu/etdc/view?acc_num=osu1307730650.
MLA Style (8th edition)
Chaiwatpongsakorn, Supranee. "Soluble Respiratory Syncytial Virus Fusion Protein in the Fully Cleaved, Pretriggered State, a Tool to Study Protein Triggering." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1307730650
Chicago Manual of Style (17th edition)
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Document number:
osu1307730650
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631
Copyright Info
© 2011, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.