Mutagenesis and Enzyme Inhibition Studies of West Nile virus NS2B/NS3 Serine Protease

Abstract

West Nile Virus (WNV) is the leading cause of mosquito‐borne disease in the United States, however there is currently no vaccine that can be used on humans to prevent or treat the infection. WNV is a +RNA virus of ~11 kbp and translated from a single open reading frame to produce a single polyprotein. This polyprotein is post‐translationally processed into three structural and seven non‐structural proteins. Specifically, NS2B and NS3 WNV proteins correspond to a serine protease which is required for further WNV viral replication (Nitsche 2018). Inhibition of this serine protease therefore represents an outstanding opportunity to possibly develop antiviral therapy. This research uses PCR to isolate a functional serine protease (NS2B/NS3), when expressed in the model bacterial organism, Escherichia coli (Shannon, 2016). An NS2B/NS2 serine protease assay will confirm the functionality of the enzyme. Utilization of known serpin (serpin protease inhibitors) will be used as positive control for the assay (Sanchez‐Navarro, AS et al., 2021). Utilization of the validated assay for NS2B/NS3 targeted mutagenesis through CRISPR/Cas9 or chemical mutagens, resulting in the identification of amino acid residues critical for functional WNV serine protease. These residues taken together with protein modelling and structure‐based design experiment, could then be used for drug development to generate novel chemical compounds or identify alternative natural product serpins (e.g. from bacteria, fungi, plants) which could ultimately prevent the virus from carrying out its final steps in replication and therefore inhibiting its ability to infect its host.