Expression and Characterization of the Powassan Virus NS2B-NS3 Protease

Introduction

While they pose little danger themselves, ticks are known hosts of many dangerous pathogens that present a severe risk to anyone bitten by them. One such pathogen is Powassan virus, an emerging, extremely deadly flavivirus found across the northern U.S. and Canada. As with other tick-borne pathogens, Powassan virus cases have increased over recent years as tick populations have grown. This virus, like all flaviviruses, relies on the NS2B-NS3 protease cleaving the non-functional polyprotein into functional protein units as an essential part of its replication cycle. Because of the necessity of this function, the protease is a very promising target for antiviral drugs treating Powassan virus, but the relatively low case numbers have led to a lack of research into the virus, and a lot of the necessary information around the protease and virus are unknown. Due to the highly fatal nature of the virus, it is important to fill this gap in knowledge before case numbers increase any further. 

In this study, we used bioinformatics tools in order to learn as much as possible about NS2B-NS3, including developing a theoretical 3D structure of the protein and predicting inhibition activity of certain known compounds. We expressed the protease in E. coli, testing both IPTG induction and autoinduction as induction methods. We purified the protein that was expressed using Ni-NTA affinity chromatography, then analyzed the resulting sample using gel electrophoresis. To validate inconsistent results, we ran further purifications using size exclusion chromatography to analyze for possible autoproteolytic activity.

Method

• Used SWISS Model to develop NS2B-NS3 3D structure based on sequence
• Tested known inhibitors’ binding with SWISS Dock
• Developed plasmid encoding 6xHis-SUMO-NS2B-NS3, lac operon, kanamycin resistance
• Transformed E. coli with plasmid
• Ran test inductions to express NS2B-NS3 using IPTG induction
• Ran large-scale induction of NS2B-NS3 expression using autoinduction
• Lysed cells, purified cell lysate with affinity chromatography
• Used ULP-1 to remove 6xHis-SUMO tag, re-ran affinity chromatography column
• Ran Size Exclusion Chromatography to test for autoproteolytic activity

Results: Bioinformatics 

• Developed 10 theoretical structures, all strong agreement around active site
• 2 distinct conformations of NS2B – open and closed
• Closed conformation had less binding activity
• Tested inhibition in both conformations of NS2B-NS3 using docking software
• Generally less negative binding energy in closed conformation, corresponding to less binding

Table 1. Binding energy of various known serine protease inhibitors on open vs closed conformations of NS2B-NS3

Results: Expression and Purification 

• Ran test inductions using IPTG induction
• Noticeable band, seems to disappear in overnight sample
• Purified protein using Ni-NTA column
• Collected highlighted fractions, ran on gel to confirm NS2B-NS3 presence
• Ran gel, noted bands below expected values, likely corresponding to autoproteolysis products.
• Ran purified sample through size exclusion column to test for autoproteolysis
• Found strong banding at expected autoproteolysis fragment sizes (19-20 kDa, 17.5 kDa, 25 kDa)

Discussion

• Developed a theoretical structure of NS2B-NS3, confirmed accuracy near active site
• Found open and closed conformations of NS2B, found that closed conformation inhibits binding to NS3
• Successfully expressed NS2B-NS3 in E. coli with both IPTG induction and autoinduction
• Successfully purified NS2B-NS3 protease, discovered and verified probable autoproteolysis

Future Directions

• Develop method to minimize autoproteolysis
• Enzyme kinetics assays with fluorescent substrate
• Inhibition tests with known protease inhibitors
• Create point mutations at specific residues to test catalytic importance
• Test conditions for crystallization to develop crystal structure