All ETDs from UAB

Advisory Committee Chair

Chad M Petit

Advisory Committee Members

Peter E Prevelige

Todd J Green

Jamil Saad

William Britt

Document Type

Dissertation

Date of Award

2019

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

The influenza A virus (IAV) non-structural protein 1 (NS1) is a highly multifunctional viral protein responsible for antagonizing the type-I interferon (IFN) response to infection. NS1 has therefore been identified as a potentially effective target for the development of novel anti-influenza compounds. Furthermore, it is important to understand the molecular underpinnings driving NS1 function to more effectively elucidate antiviral targets. In this dissertation, we have contributed significant insight into NS1’s potential as an antiviral target, and the structure-function relationships driving its activity in an infected cell. First, we structurally characterized the binding of two known influenza inhibitors (A9 and A22) to the NS1 effector domain (NS1ED). Using nuclear magnetic resonance (NMR) chemical shift perturbation (CSP), we identified the A9/A22 binding site on the NS1ED. The location of the binding site overlapped precisely with the hydrophobic pocket in which the 30 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) binds to the NS1ED (CPSF30 binding pocket), suggesting that A9/A22 block this critical interaction. Additionally, we observed A9/A22 binding to the NS1ED of two highly pathogenic strains of IAV, indicating potential broad specificity of these compounds. Next, we solved the structures of the A/Brevig Mission/1/1918 (1918H1N1) NS1ED wild-type (1918H1N1 NS1ED WT) and D171A mutant (1918H1N1 NS1ED D171A) using X-ray crystallography. In comparing these structures, we identified a novel intramolecular electrostatic interaction that was only present in the 1918H1N1 NS1ED WT structure. The loss of this interaction upon introduction of the D171A mutation was correlated with altered flexibility in two highly variable loop regions (140- & 170-loops) and an increase in solvent exposure in the 170-loop. Furthermore, the D171A mutation caused a decrease in CPSF30 binding affinity. Conversely, the reciprocal A171D mutation, introduced into a recombinant IAV NS1 (rPR8A171D), increased replication in A549 cells and in primary normal human bronchial epithelial (NHBE) cells compared to a wild-type recombinant IAV (rPR8WT). rPR8A171D also increased antagonism of the type-I IFN response and nuclear retention of NS1 in infected cells. Finally, rPR8A171D exhibited increased pathogenicity in C57BL/6 mice compared to rPR8WT. Taken together, these data generate important insights into NS1 structure, function, and potential as an antiviral target. Furthermore, this work brings into sharper focus numerous open questions regarding NS1 multi-functionality and provides a foundation upon which future studies can be built.

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