Date of Award

May 2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

David N Frick

Committee Members

Valerica Raicu, Alexander Arnold, Doug Steeber, Nick Silvaggi

Keywords

FRET, FRET Spectrometry, HCV, LGP2, NS3, RIG-I

Abstract

The innate immune system is a part of the first line of defense against virus infection. An important subset of the innate immune system consists of a group of intracellular pattern recognition receptors (PRRs) which recognize conserved features of bacteria and viruses and initiate an interferon response. The RIG-I like receptors (RLRs) are PRRs that bind to RNA viruses (such as hepatitis c virus) and signal through the adaptor mitochondrial anti-viral signaling protein (MAVS).

Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the flaviviridae family of viruses. HCV infects hepatocytes and can cause a persistent infection. If a chronic infection is established, progressive liver damage along with cirrhosis and sometimes hepatocellular carcinoma may occur. The multi-functional HCV non-structural-3 (NS3) protein is essential for HCV replication and contains covalently linked protease and helicase/ATPase domains. A covalently linked protease and helicase is unique to the flaviviridae family of viruses and it is unclear why the two functions are linked. There are multiple effective direct acting anti-virals which target the protease, but none currently approved which inhibit the helicase. In addition to aiding in viral replication, the NS3 protease assists HCV in establishing a persistent infection through cleaving the innate immune RIG-I adaptor protein, MAVS. The purpose of the studies contained in this thesis is to gain a greater understanding of the function and purpose of the covalently linked HCV NS3 protease and helicase. Förster resonance energy transfer (FRET) is used to explore the interaction of NS3 with RIG-I like receptor proteins and to use FRET to look at the interaction of the RLRs with themselves.

The interaction of the NS3 protease and helicase domain was probed through the exploration of the mechanism of action of a NS3 inhibitor (HPI) which is able to inhibit both the protease and helicase functions of NS3, while not disrupting the ATPase activity. The activity of HPI was determined in vitro using a fluorescent protease cleavage assay and a fluorescent helicase unwinding assay. HPI can inhibit both functions with low micro-molar EC50. Next, analysis of HPI to inhibit peptide hydrolysis by wild-type NS3 and a set of NS3 mutants with mutations in the protease domain, helicase domain, and the allosteric groove between the protease and helicase domain suggested that HPI forms a bridge between the NS3 helicase RNA-binding site and the allosteric groove between the protease and helicase domains. The activity of HPI was measured in cells using an HCV sub-genomic replicon tagged with a luciferase reporter. The inhibition of HPI alone and in the presence of other protease inhibitors was tested. HPI can inhibit the HCV genotype 1b sub-genomic replicon and when applied in conjunction with first generation protease inhibitors, telaprevir and boceprevir, the inhibition was additive, as defined by the Bliss Independence Model of additive inhibition. However, when HPI was used in conjunction with macro-cyclic protease inhibitors, danoprevir and grazoprevir, modest synergy was observed.

To look at the protein:protein interactions of the NS3 helicase and the RIG-I like receptor helicases in live cells, a series of quantitative FRET spectrometry studies were employed. Quantitative micro-spectroscopic imaging (Q-MSI) is a technique which uses a fluorescent dye or fluorescent protein to identify sub-cellular regions and then calculates Förster Resonance Energy Transfer (FRET) efficiency and the concentrations of the donor and acceptor proteins. The technique was first applied in vitro with a fluorescently tagged NS3 helicase and fluorescently tagged DNA molecules. Next, the technique was applied to combinations of recombinant fluorescently tagged helicases expressed in HEK293T cells. The NS3 helicase, RIG-I like receptor helicases, DDX1, DDX3, and DDX5 helicases, and MAVS were all designed to express off plasmids which also encode and attach a fluorescent protein. The fluorescent proteins used were either cyan fluorescent protein (CFP), enhanced green fluorescent protein-2 (GFP2), yellow fluorescent protein (YFP) or Venus fluorescent protein and each combination included a donor (CFP or GFP2) and an acceptor (YFP or Venus) fluorescent protein. The combinations were tested in presence or absence of polyinosinic-polyctyidlic acid (poly I:C) which is a synthetic RNA analog capable of eliciting an RLR response. To localize the interaction to the mitochondria, the mitochondrial stain, Mito-Tracker-Red, was used in some experiments.

The experiments revealed a previously unknown interaction between NS3 and the RLR protein, laboratory of genetics and physiology protein-2 (LGP2) which may be biologically relevant. In addition, the relocation of LGP2 cytoplasmic foci in cells over-expressing DDX3 was observed. Q-MSI was used to visualize previously known interactions of RLRs at the mitochondria and in conjunction with MAVS.

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