Dual Perspectives of Innate Immunity: Investigation of Interferon Signaling Requirements & Characterization of Antagonism by RNA Viral Proteases

Abstract

Humans and viruses have long engaged in an evolutionary arms race. As cells detect viruses and produce interferons (IFNs) to stop them, many viruses utilize their virally encoded proteases to impair IFN production, among various other strategies. The aims of this thesis were twofold. First, the way in which multiple type I or III IFN subtypes elicit different signaling outcomes via their respective receptors is poorly understood. We investigated the requirement for tyrosine kinase 2 (TYK2) in type I and III IFN signaling by testing the ability of IFN subtypes to signal in CRISPR/Cas9-mediated TYK2 knockout cells. We found that TYK2 was dispensable for type III IFN signaling but was differentially required across type I IFN subtypes. To investigate mechanisms by which IFN signaling occurs in the absence of TYK2, we built chimeric IFN receptors capable of homodimerizing. We observed that homodimers of IFNAR2 (type I) or IFNLR1 (type III) phosphorylated STAT1 to different levels. Our results suggest that utilization of TYK2 and differences within the receptor subunits themselves may contribute to differential type I and III IFN signaling. The second part of this thesis aimed to characterize how the hepatitis E virus (HEV) open reading frame 1 (ORF1) protein harboring a putative protease domain antagonizes the innate immune system by comparing it to the known antagonizing abilities of proteases of several other RNA viruses. To do so, three pathways of IFN induction were stimulated in the presence of HEV ORF1 or other RNA viral proteases, and the expression of IFNs and IFN-stimulated genes was measured by RT-qPCR. We found that HEV ORF1 exhibited broad innate immune antagonism and the virally encoded proteases of West Nile virus, dengue virus, and hepatitis C virus demonstrated both expected and novel methods of antagonizing the innate immune system. This work not only contributes to a more complete understanding of HEV biology, but the study of innate immunology and its applications to virology also provide new avenues for potential broad-acting therapeutics against existing and emerging viruses.