Dissertation/Thesis Abstract

Structure-function characterization of the role of Epstein-Barr virus glycoprotein B in fusion
by Reimer, Jessica Jean, Ph.D., Northwestern University, 2009, 195; 3386956
Abstract (Summary)

Glycoprotein B is one of the most functionally conserved proteins in the Herpesvirus family, and is an essential component of the entry machinery for most human Herpesviruses. EBV gB is required for fusion in cell-based assays with both epithelial and B cells, the main target cells of EBV in vivo infection. To further characterize the role of gB in fusion, two distinct studies were performed: targeted mutagenesis of a proposed epithelial cell receptor-binding domain and random linker insertion mutagenesis to identify other possible functional domains.

Three mutants were constructed by altering conserved residues of the proposed epithelial cell receptor-binding region, the disintegrin-like domain. Only one of the three mutants was processed to the mature form of gB, while none of the mutants were capable of functioning in fusion with epithelial or B cells. Careful study of the EBV gB crystal structure shows that this region shares no structural homology with known integrin-binding proteins. In addition, this region is not accessible to solvent in the pre-fusion or post-fusion gB structures, indicating that it is unlikely to engage receptor.

The linker insertion study produced several contributions to the understanding of EBV entry. In total, 23 linker insertion mutants were examined for altered processing, expression and function in fusion. A N-glycosylated mature form of gB was discovered that is a critical determinant of function in fusion. Only three mutants (gB30, gB421 and gB801) were processed to the mature form; they were fully functional in fusion with epithelial cells. These mutants were further characterized in a B cell viral entry assay. Of these three mutants, gB30, located in the N-terminus of the protein, exhibited significantly reduced membrane fusion ability with B cells and was unable to complement gB-null virus in the viral entry assay. This points to a previously unidentified function of gB in B cell fusion, possibly regulating tropism through interaction with another viral glycoprotein or B cell-specific cellular component.

In combination, the mutagenesis of EBV gB performed herein and the subsequent structural analysis provide additional support for the essential role of gB as the EBV fusogen.

Indexing (document details)
Advisor: Longnecker, Richard
Commitee: Jardetzky, Theodore, Smith, Greg A., Spear, Patricia G.
School: Northwestern University
Department: Integrated Graduate Program in the Life Sciences
School Location: United States -- Illinois
Source: DAI-B 70/12, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Microbiology, Virology
Keywords: Epstein-Barr virus, Glycoprotein B, Herpesvirus, Linker insertion mutagenesis, Viral entry, Viral fusion
Publication Number: 3386956
ISBN: 9781109528077
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