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Costello Dissertation 20130815.pdf (3 MB)

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The N500 Glycan of the Respiratory Syncytial Virus F Protein is Required for Fusion, but Not for Stabilization or Triggering of the Protein

Costello, Heather M
http://rave.ohiolink.edu/etdc/view?acc_num=osu1376568814

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Integrated Biomedical Science Graduate Program.
Respiratory syncytial virus (RSV), a paramyxovirus, is the most significant respiratory pathogen in infants and causes 90,000 emergency hospitalizations in the United States and 160,000 deaths worldwide every year. It is also a leading respiratory pathogen in the elderly. RSV infection provides weak immunity, and the virus infects individuals repeatedly throughout life. The only effective antiviral compound commercially available is a monoclonal antibody, palivizumab (Synagis®), which is given prophylactically to at-risk infants. Despite intense efforts, no other therapeutic and no vaccines have been approved for use, although several are in development. Most drugs developed against RSV target the fusion (F) glycoprotein. The F protein is responsible for fusing the host cell and viral membranes together to initiate infection. The RSV F protein is unique among other paramyxoviruses in that it does not require its partner attachment (G) glycoprotein to function as do most other paramyxovirus F proteins. It is the subject of extensive research in hopes of determining its mechanism of action and developing anti-RSV compounds. The mature, cleaved RSV F protein contains N-glycans at asparagine residues 27, 70, and 500. It has previously been reported that none of these N-glycans are required for protein processing or cell surface expression, but that the N500 glycan is required for cell-to-cell fusion in an assay serving as a proxy for virion-cell fusion. In this study, we replicated these results in the full-length protein. We also built N27Q, N70Q, N500Q, N27/70Q, and N/27/70/500 mutations into a soluble version of the RSV F protein to determine the role, if any, that these N-glycans have in triggering. Following purification, each of the N-glycan mutants migrated near the top of a sucrose velocity sedimentation gradient, similar to the wild type (WT) sF protein, indicating that these proteins were produced and secreted from the cell in the prefusion form. We determined that none of these glycans were responsible for maintaining the prefusion form, and that premature triggering is not the reason for the fusion deficiency of the N500Q mutant. We have previously determined that exposure to low molarity triggers the RSV F protein, resulting in the exposure of its hydrophobic fusion peptide and resultant aggregation. Following dialysis of the WT and mutant sF proteins into a 50 mM Hepes buffer, each migrated to the bottom of a sucrose velocity sedimentation gradient, indicating that the proteins had triggered and aggregated by their hydrophobic fusion peptides. All of the N-glycan sF proteins were triggerable, indicating that the inability of the N500Q mutant to fuse is not the result of an inability to trigger and is likely due instead to an inability to refold properly following triggering. The N500Q glycan is attached to the HRB domain of the RSV F protein and may guide the HRB as it completes formation of the 6-helix bundle, a characteristic form of the postfusion F protein that brings the virion and cell membranes into close proximity and results in the fusion of these two membranes.
Mark Peeples, Ph.D. (Advisor)
161 p.
Biomedical Research; Virology
respiratory syncytial virus; fusion protein; paramyxovirus; viral entry; antiviral; vaccine; N-glycan

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Citations

  • Costello, H. M. (2013). The N500 Glycan of the Respiratory Syncytial Virus F Protein is Required for Fusion, but Not for Stabilization or Triggering of the Protein [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376568814

    APA Style (7th edition)

  • Costello, Heather. The N500 Glycan of the Respiratory Syncytial Virus F Protein is Required for Fusion, but Not for Stabilization or Triggering of the Protein. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1376568814.

    MLA Style (8th edition)

  • Costello, Heather. "The N500 Glycan of the Respiratory Syncytial Virus F Protein is Required for Fusion, but Not for Stabilization or Triggering of the Protein." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1376568814

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.