All ETDs from UAB

Advisory Committee Chair

Margaret A Johnson

Advisory Committee Members

Aaron L Lucius

William J Placzek

Jamil S Saad

Sadanandan Velu

Document Type

Dissertation

Date of Award

2018

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

Since 2003, two severely human pathogenic viruses have emerged as a formidable health threat with no effective treatment to date. These viruses belong to the coronavirus (CoV) family. Upon infection, this viral family uses host cell machinery to translate nonstructural proteins (nsps) which are used to facilitate the viral life cycle. The first major human viral threat, the severe acute respiratory syndrome (SARS) virus, contains a multidomain region within its nsp3 called the SARS Unique Domain (SUD). This region contains macrodomains, which are implicated in viral replication. Macrodomains bind monomeric and polymeric ADP-ribose, a biomolecule involved in many cellular processes. Our laboratory has focused on determining the structure of viral macrodomains to investigate how their structures are related to function. We employ this strategy by studying macrodomains from bat CoVs. Since bats are a main progenitor host of human viruses, shared conserved features provide insight into how these domains evolve to infect humans. We developed and optimized protocols for the expression of recombinant proteins and analyzing the samples via bioinformatic analysis and computational docking programs, nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC), circular dichroism (CD), and mass spectrometry (MS). Using solution nuclear magnetic resonance (NMR) to determine protein structure and other spectroscopic and calorimetric methods, we studied SUD homologs in the bat CoVs Tylonycteris pachypus (HKU4) and Rousettus (HKU9). We are able to show the SUD domain is not limited to the SARS virus but is located in other viruses. The macrodomain in HKU4 acts as an enzyme to remove ADP-ribose modifications from protein substrates. When we mutated specific residues in the binding site, we found that both ligand binding and enzymatic activity were affected. Finally, we solved the structure of an HKU9 accessory domain, the C domain. We showed the C domain has a highly conserved and globular fold to the SARS C domain. These similarities indicate the C domain of bat CoVs has a shared function with respect to human CoVs. Overall, the findings described here expand our understanding of viral macrodomain diversity and the structural features that affect its activity.

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