Functional and structural characterization of Flaviviral NS5 replication machinery : study of Powassan virus NS5 a tick-borne virus

• Main Author: Chang, Melissa Hui Min
• Other Authors: Julien Lescar
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/139884
• Institution: Nanyang Technological University

Powassan virus (POWV) is a re-emerging old world flavivirus that is well understudied despite being genetically sequenced three decades ago. Recent epidemiological data has shown the need to generate potential inhibitors against this neuro-invasive virus. Non-structural protein 5 (NS5) is one of the most conserved protein as well as an essential replication enzyme in the replication complex. Targeting NS5 has generated multiple potential inhibitory compounds in other flavivirus and could be adapted for POWV. Unfortunately, there is a severe lack of structural and functional studies on NS5 in tick-borne flavivirus, let alone POWV. Furthermore, due to the lack of NS5-RNA complex structure, NS5 was never studied beyond the pre-initiation stage. This paper was the first to produce high purity POWV NS5 through the adaptation of bacterial expression system and purification process. Further investigation of POWV NS5 showed that while the sequence was generally conserved, the linker region was not. POWV NS5 also demonstrated low affinity for RNA, hampering efforts to obtain a structure for the NS5-RNA complex. This work paves the way for the structural and biophysical study of POWV NS5 and participates in the effort to obtain structural information of the active replicative NS5-RNA complex.