Single molecule investigation of antiviral signaling

Retinoic acid-inducible gene 1 (RIG-I) plays a major role in antiviral innate immunity by detecting cytoplasmic viral RNA and triggering the pathway that leads to transcriptional activation of type I interferon (IFN). Type I IFN induces a large set of genes called interferon-stimulated genes (ISGs) that coordinate a response to antagonize viral invasion. We utilized single-molecule fluorescence in situ hybridization (smFISH) to study viral and antiviral gene expression in individual cells. We precisely quantified kinetics of mRNA expression of RIG-I with respect to IFN- in cells upon infection with Sendai virus (SeV). We found that RIG-I mRNA is directly induced by viral infection in the absence of IFN at the early stages of viral infection. High throughput screening of ISGs expression following viral infection revealed other genes, which we termed as “early genes”, induced via the IFN-independent mechanism. We found that this mechanism is dependent on interferon regulatory factor 7 (IRF7) and interferon regulatory factor 3 (IRF3). Simultaneous detection of mRNA of IFN-, viral L gene, and ISGs revealed distinct populations of IFN- expressing and non-expressing cells which are dependent on the early genes but are independent of other ISGs and the viral load. Single-cell analysis of IRF7/3- and NF-kB-dependent gene expression suggest that early IRF7/3-dependent gene expression may facilitate the activation of transcription factor NF-kB and contribute to the decision making process for IFN- production. Fluorescence microscopy is an essential tool in biology. However, fluorescence microscopy is limited by diffraction to a spatial resolution of >200 in lateral dimensions, and >500 nm in axial dimension, leaving many biological structures too small to study in detail. With the recent developments in super-resolution imaging techniques, the resolution limit of conventional fluorescence microscopy has been surpassed. One such technique is referred to as stochastic optical reconstruction microscopy (STORM), and provides ~ 20 nm lateral and ~ 50 nm axial resolution. We constructed a STORM microscope, and used it to image cellular localization of RIG-I in the virally infected cells. STORM imaging revealed clustering of RIG-I, that was not visible under conventional fluorescence microscope. One of the critical aspects of a virus life cycle is packaging of the viral genome. We studied packaging mechanism of Influenza A Virus (IAV). IAV possesses a segmented genome of eight, single-stranded RNAs. However, the exact copy number of each viral RNA segment per individual virus particle has been controversial for the past 50 years. To address this question, we combined single molecule TIRF microscopy and fluorescence in situ hybridization (FISH) to study the composition of viral RNAs at single-virus particle resolution. Our results showed that a high percentage of virus particles package a single copy of each segment of viral RNAs. Our findings support a model that the packaging of IAV genome is a selective and robust process.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Computnl Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2014

Author dc:creator

• Doganay, Sultan

Contributors dc:contributor

• Ha, Taekjip
• Belmont, Andrew S.
• Tapping, Richard I.
• Schroeder, Charles M.

• Retinoic acid-inducible gene 1 (RIG-I)
• interferon beta (IFNB)
• antiviral signaling
• single molecule fluorescence in situ hybridization

Statement dc:rights

• Copyright 2013 Sultan Doganay

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/46595

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/46595