Doctor of Philosophy in Pathology and Laboratory Medicine (PhD) 
Roles of microRNAs in Coxsackievirus B3 induced viral myocarditis
Arbutus Biopharma Inc.
Viral myocarditis is an inflammatory heart disease caused by viral infection, which is a major cause of sudden death in children and young adults. Among the various viruses, coxsackievirus B3 (CVB3) is a predominant pathogen of viral myocarditis. As CVB3 replication is tightly tangled with signaling pathways in host cells, an in-depth study of CVB3-host interactions would promote the understanding of the pathogenesis of viral myocarditis and provide critical drug targets for the development of therapeutics. CVB3 infection induces different types of stress in host cells, and in turn, the cells respond to the stress via expressing certain stress-responsive proteins (SRPs) to counteract the stress for cell survival. During the co-evolution of virus and host, CVB3 has developed sophisticated strategies to modulate and utilize SRPs to benefit its own replication. The main objective of this dissertation is to investigate the modulation and functional roles of SRPs in CVB3 infection and CVB3-induced myocardium damage. I hypothesize that 1) CVB3 infection differentially regulates the expression and activity of SRPs at transcription, translation or post-translation level; 2) the dysregulation of SRPs benefits CVB3 replication and promotes CVB3-induced cell damage. This dissertation mainly focuses on two SRPs, the inducible heat shock 70 kDa protein (Hsp70) and nuclear factor of activated T-cell 5 (NFAT5), during CVB3 infection. Using in vitro (cell culture) and in vivo (mouse) models, I demonstrated an increase of Hsp70 but a decrease of NFAT5 during CVB3 infection. Further studies elucidated the mechanism underlying such changes as well as the feedback effects on CVB3 replication. Hsp70 was upregulated via CaMKII-HSF1 signaling cascade activated in CVB3 infection and in turn promoted CVB3 infectivity via stabilizing viral genome and benefiting viral translation. NFAT5 was cleaved by CVB3 proteases 2A and 3C, generating a 70 kDa dominant negative truncate, which inhibited the iNOS-mediated anti-viral activity of NFAT5. Together, my findings have uncovered the new roles of SRPs in CVB3 infection and potential novel drug targets for CVB3-induced myocarditis.
Myocarditis, inflammation of the heart muscle, is a spectrum of conditions causing significant morbidity and mortality, yet scientific and clinical knowledge related to this entity is limited. One of the most common and best studied causes of myocarditis is infection by coxsackievirus B3 (CVB3). An improved understanding of the science behind CVB3 myocarditis is critical to establishing better diagnostic and therapeutic strategies for affected individuals. CVB3 infection redirects numerous cellular pathways from physiologic processes to viral replication, often mediated by viral proteases. Two viral targets in this process are death associated protein 5 (DAP5) and nuclear pore complex protein 98 (Nup98). DAP5 is a translation initiation factor specific to internal ribosome entry site (IRES) mediated translation. Nup98 is a component of the nuclear pore complex and a transcription factor.In this thesis, I hypothesize that viral proteases contribute to the pathogenesis of viral myocarditis through interaction with DAP5 and Nup98, redirecting translation and transcription towards viral replication.Using in vitro (plasmid expressed viral proteases), in situ (CVB3 infection in cell culture), and in vivo (mouse myocarditis model) models, I demonstrate that viral protease 2A is responsible for the cleavage of DAP5 and Nup98 during CVB3 infection. Both cleavage events I show to be integral to the viral lifecycle using over expression of recombinant fragments and siRNA inhibition of that expression.These results suggest two previously unidentified targets for improved diagnostics and therapeutics for myocarditis, both areas for future research.
Coxsackievirus B3 (CVB3) induced viral myocarditis, characterized by inflammation and cell death in the myocardium, is one of the leading causes of sudden unexpected death in infants and young adults. Both direct virus- and immune-mediated injuries contribute to the damage in the infected organs. A clear understanding of the virus-induced host cell signaling alterations would be key to elucidating the pathogenesis of viral myocarditis and to improve therapeutic strategies. Recently discovered microRNAs (miRNAs) are small endogenous non-coding RNAs widely involved in gene regulation controlling developmental processes and disease pathogenesis including cardiac diseases and viral infections. In this dissertation, the main objective is to investigate the roles of miRNAs in CVB3 replication and pathogenesis of myocarditis. I hypothesize that 1) CVB3 infection alters host miRNA expression profiles to benefit its own replication; and 2) the dysregulated miRNAs contributes to the damage and dysfunction of cardiomyocytes. I used in vitro (cultured cells) and in vivo (mouse) models to explore the changes of host miRNAs’ expression during CVB3 infection. miRNA microarray and quantitative-reverse transcription-PCR (q-RT-PCR) revealed that miR-126, miR-203 and miR-21 were upregulated by CVB3 infection. Further studies on these three miRNAs demonstrated their unique roles in regulating viral replication and cellular pathology in the myocardium. miR-126 was induced by CVB3 infection through the ERK1/2-ETS1/2 signal pathway. I found that increased miR-126 in turn enhanced activation of ERK1/2 and degradation of β-catenin through targeting SPRED1, LRP6 and WRCH1. This targeting benefited CVB3 replication and promoted virus-induced cell death. miR-203 was upregulated by the activation of the PKC/AP-1 cascade during CVB3 infection. I showed that miR-203 targeted ZFP-148 and supported cell survival and growth, which provided favorable environment for CVB3 replication. I further conducted the first investigation on the role of miR-21 in cell-cell connections among cardiomyocytes during CVB3 infection. I showed that miR-21 upregulation induced desmin degradation and desmosome disorganization via ubiquitin-proteasome pathway by targeting YOD1 and that miR-21 directly targeted VCL and disrupts fascia adherens. Together, my findings have shed light on the host-virus interactions in signal transduction pathways and provided new therapeutic strategies against CVB3-induced heart diseases.
Interferon-γ-inducible GTPase (IGTP, or IRGM3) is a p47 GTPase upregulated in coxsackievirus B3 (CVB3)-infected murine heart and inhibits CVB3-induced apoptosis through activation of the PI3 kinase/Akt pathway. However, the mechanism of this PI3K/Akt activation and other prosurvival functions of IGTP are unknown. In this study, using a doxcycycline-inducible Tet-On HeLa cell line that overexpresses IGTP, I have demonstrated that focal adhesion kinase (FAK) is phosphorylated in response to IGTP expression and that transfection of a dominant negative FAK (FRNK) blocks Akt activation. Furthermore, induction of IGTP promoted the NF-κB activation as evidenced by its enhanced nuclear translocation and increased transcriptional activity. However, FRNK transfection and PI3K inhibitor both blocked the IGTP-induced translocation and NF-κB activation. Moreover, silencing NF-κB with siRNAs significantly inhibited the phosphorylation of FAK and Akt. Finally, blocking this survival pathway by FRNK transfection or NF-κB siRNA reduced CVB3 replication and enhanced cell death during CVB3 infection. Taken together, these results suggest that FAK is a mediator upstream of PI3K/Akt and that NF-κB functions as a downstream effector. As viral infections including CVB3 can cause endoplasmic reticulum (ER) stress and activate a group of coordinated signal pathways termed ER stress response, additional prosurvival mechanisms of IGTP related to ER stress response was explored. It was demonstrated that IGTP expression suppressed either chemical- or CVB3-induced upregulation of GRP78, the ER stress marker. IGTP expression strongly inhibited the activation of both the PERK and ATF6 pathways of ER stress responses. The suppression of ER stress responses by IGTP also led to attenuated induction of proapoptotic genes CHOP and GADD34. These data were further supported by experiments using IGTP knockout mouse embryonic fibroblast cells, in which the ER stress response induced by CVB3 infection was not relieved after interferon-γ treatment due to the absence of IGTP. Moreover, blocking PI3K/Akt pathway with either the PI3K inhibitor or dominant negative Akt construct significantly diminished the inhibitory effect of IGTP on ER stress response as well as its prosurvival effect. Therefore, IGTP expression relieves the ER stress response via a PI3K/Akt dependent mechanism, which contributes to cell survival and host defense during CVB3 infection.
No abstract available.