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Molecular and cellular studies of the West Nile virus NS2B/NS3 protease

University of British Columbia

West Nile virus (WNV) is the most widely distributed arthropod-borne virus globally. It can cause a potentially fatal infection and has become a public health concern in North America since its introduction in 1999. Currently, there are no vaccines or treatments available for human WNV infections. As such, it is important to understand the virus life cycle, in order to develop effective therapeutics. The WNV protease heterocomplex, NS2B/NS3, is a prime target for antiviral therapy and has become the focus of much research. It is important to understand protease function first, in order to develop effective inhibitors. The overall goal of this thesis was to gain a better understanding into the function of the full-length NS2B/NS3 protease heterocomplex within the intracellular microenvironment. I hypothesized that there are critical residues essential for the interaction between NS2B and NS3 that affect protease activity and protein stability. The first aim of this project was to generate a cell-based fluorescent substrate assay to investigate the protease activity of the full-length NS2B/NS3 protease heterocomplex within the cell. My results demonstrate that the full-length NS2B/NS3 protease heterocomplex functions differently within the context of the cell, compared to what has been previously observed in vitro (Chapter 2). In the second aim, I investigated NS2B function on NS3 protease cis-cleavage and trans-cleavage activity. My results reveal an important dual role the NS2B protein plays in the proper function of the full-length NS2B/NS3 protease heterocomplex (Chapter 3). In the third aim, I utilized the information gathered to rationally design and test a serine protease inhibitor directed against the full-length NS2B/NS3 protease heterocomplex (Chapter 4). Taken together, my results highlight the importance of utilizing cell-based assays to assess protease activity, as this allows for the investigation of NS2B/NS3 protease function in a more physiologically relevant environment. The results presented in this thesis further our understanding of the activity of the full-length WNV NS2B/NS3 protease heterocomplex within the context of the cell. The information gathered gives insight into the regulation of viral protease function that could be utilized in the rational drug design towards the WNV NS2B/NS3 protease heterocomplex.

Text

2010-07-29

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/26994

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/