Degree Type

Dissertation

Date of Award

2014

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Gaya K. Amarasinghe

Abstract

Viral protein 35 (VP35), encoded by filoviruses, are multifunctional dsRNA

binding proteins that play important roles in viral replication, innate immune evasion and pathogenesis. The multifunctional nature of these proteins also presents opportunities to develop countermeasures that target distinct functional regions. However, functional validation and the establishment of therapeutic approaches toward such multifunctional proteins, particularly for non-enzymatic targets, are often challenging. With the current lack of approved vaccines or therapeutic options available to target filoviral infections, work that lends itself to the development of such inhibitors will be instrumental in countering this highly pathogenic virus. Our previous work on filoviral VP35 proteins defined two highly conserved conserved basic patches, the first basic patch (FBP) and the central basic patch (CBP), located within the C-terminal dsRNA binding interferon (IFN) inhibitory domain (IID). This work went on to show that the CBP is important for VP35 mediated IFN antagonism, in part through its ability to bind dsRNA. The goal of my thesis work was to investigate the functional importance of the FBP through a combination of structural and biochemical studies, and validate VP35 as a potential therapeutic target. These efforts established that residues within the FBP are functionally distinct from the CBP, but are important for VP35 polymerase co-factor function. Although the exact role of these VP35 residues in replication is poorly defined, the replication defective FBP mutants lost their ability to interact with the viral nucleoprotein, indicating that the VP35 FBP provides critical contacts which establish the VP35-NP interaction. In order to therapeutically target these functional regions of VP35 and validate VP35 as a potential and promising antiviral target, we targeted Ebola virus (EBOV) VP35 (eVP35) for aptamer selection using SELEX. Select aptamers, representing two distinct classes, were further characterized based on their interaction properties to eVP35 IID. These results revealed that the aptamers bind to distinct regions of eVP35 IID with high affinity (10-50 nM) and specificity. These aptamers can compete with dsRNA for binding to eVP35 and disrupt the eVP35-NP interaction. Consistent with the ability to antagonize eVP35-NP interaction, select aptamers can inhibit the function of the EBOV polymerase complex reconstituted by expression of select viral proteins. Taken together, our results serve as an initial step in enhancing our understanding of the protein-protein interactions that establish the EBOV polymerase complex, and support the identification of two aptamers that bind filoviral VP35 proteins with high affinity and specificity and have the capacity to potentially target filoviral VP35 proteins as a therapeutic target.

Copyright Owner

Jennifer Binning

Language

en

File Format

application/pdf

File Size

152 pages

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