All ETDs from UAB

Advisory Committee Chair

Chad M Petit

Advisory Committee Members

Troy Randall

Kevin Harrod

William Placzek

Todd Green

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The influenza virus non-structural protein 1 is well known to antagonize the host innate immune response through its interaction with the innate immune sensor, retinoic acid induc-ible gene I (RIG-I). While the complete mechanism of the NS1:RIG-I interaction remains unclear, we were the first to demonstrate a direct interaction between the NS1 RNA-binding domain (NS1RBD) from the 1918H1N1 influenza virus and the second caspase activa-tion and recruitment domain (CARD2) of RIG-I using NMR. In addition, we also identi-fied that mutation of Arg 21 in the 1918H1N1 NS1RBD to Gln (R21Q) completely abrogated the NS1:CARD2 interaction. Given that CARD2 plays a critical role in RIG-I activation through its ubiquitination and that wild-type NS1 is known to inhibit the ubiquitination of CARD2 during infection, we hypothesized that the R21Q mutation in NS1 may result in less efficient antagonism of CARD2 ubiquitination. To determine the role that residue 21 in NS1 plays during infection, we constructed recombinant PR8 influenza viruses with WT and R21Q-mutant NS1s using reverse genetics (rPR8WT and rPR8R21Q). We found that during infection, rPR8R21Q results in significantly increased IRF3 phosphorylation and IFN-β induction suggesting that rPR8R21Q is a more potent activator of RIG-I signaling. This data is supported by overexpression studies where we determined that R21Q mutant NS1 (NS1R21Q) is significantly less efficient in preventing TRIM25-mediated ubiquitination of the RIG-I CARDs when compared to WT NS1 (Ns1WT). Additionally, we confirmed that the molecular basis behind these differences is indeed due to NS1R21Qs inability to in-teract with the RIG-I CARDs in a TRIM25-independent manner. These findings demon-strate that the R21Q mutation in the NS1RBD results in increased RIG-I activation and type I interferon induction due to deficient antagonism of ubiquitination of the RIG-I CARDs. Consequently, viruses with the R21Q mutation may be less virulent than strains containing an Arg at position 21 in the NS1 protein. These novel findings are yet another example of structural biology informing on overall protein function that we hope will aid in future sur-veillance of emerging strains and the discovery of novel therapeutics for the influenza virus.

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