Structural and biochemical characterization of non-structural protein 3 from flavivirus
Phoo, Wint Wint
Date of Issue2018-12-03
School of Biological Sciences
Lee Kong Chian School of Medicine
Flaviviruses are positive sense RNA viruses transmitted by arthropods. The flavivirus genus includes many important human pathogens such as Dengue Virus (DENV), West Nile virus (WNV), Yellow fever Virus (YFV), and recently emerged Zika Virus (ZIKV). About 40% of the world population is at risk of the flavivirus infections. The latest outbreaks of ZIKV in 2015 resulted in World Health Organization declaring Public Health Emergency of International Concern (PHEIC). There are neither specific nor broad-spectrum antivirals against flavivirus infections. The flavivirus polyprotein encodes three structural and seven non-structural proteins. Non-structural protein 3 (NS3) is the second largest protein encoded by the virus and composed of two domains, N-terminal protease and C-terminal RNA helicase/adenosine triphosphatase (ATPase) domains. The enzymatic activities of nonstructural protein 3 are essential for viral RNA replication in the host cell making NS3 a prime target for the design of compounds with antiviral activity. In this study, 3D X-ray crystallography structures of NS2B-NS3 protease from ZIKV unbound and bound to different inhibitors are reported. The structures of ZIKV protease with different inhibitors compounds contributes significantly to the compound optimization in structure-based drug discovery. Biochemical assays were also conducted to characterize the NS2B-NS3 protease activity with and without inhibition by different compounds. In addition, structural and biochemical properties of the NS2B-NS3 protease and NS2B-NS3 full length from DENV, a closely related flavivirus, were studied. The structure of NS2B-NS3 protease from DENV reveals a new conformation which could be useful in virtual screening with compounds. We reported free enzyme structures and NS2B-NS3 complex in complex with bovine pancreatic trypsin inhibitor (BPTI). Altogether, the work in this thesis provides insights into the dynamics of the protease domain in full length context as well as conclusive biochemical activities of each domain from full length NS3 protein.