Functional characterization of oligoadenylate synthetase (OAS) by dual polymerase-endoribonuclease assay
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The innate immune system includes a class of viral double stranded RNA (dsRNA) binding enzymes known as 2’-5’-oligoadenylate synthetases (OAS). The OAS family detects viral dsRNA and initiates downstream processes. The binding of viral dsRNA by OAS enables catalysis of substrate ATP into 2′-5′-linked oligoadenylate chains (2-5A). 2-5A chains longer than 3 nucleotides then activate an RNA degrading enzyme named RNase L, which in turn non-specifically degrades cellular and viral RNA causing host cell death. My research group has been investigating the interaction between the smallest OAS family member, OAS1 (42 kDa), with a double-stranded region of the West Nile virus (WNV) RNA genome. This region of the genome is conserved amongst the Flaviviridae family of viruses, of which WNV is a member. Therefore, the goal of my work is to see whether similar RNA regions in other Flaviviridae members using Zika Virus, Japanese Encephalitis Virus, WNV and Dengue Virus as model systems for studying this interaction. The approach will be to produce viral RNA from the conserved regions and determine the minimal RNA required for binding of OAS1 and catalytic activation of OAS1. I have produced a dual OAS-RNase L activity assay which provides data in shorter time intervals, measures OAS activity and RNase L activity simultaneously, and requires very low concentrations of reactants. The data obtained from this assay will be used to assess activation of OAS enzyme by flavivirus RNA and help paint a more complete picture of OAS mechanism of action.