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PublisherThe University of Arizona.
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AbstractRhinovirus (RV) and influenza virus infections are the leading causes of airway tract problems that lead to cold and flu and exacerbated asthma, chronic obstructive pulmonary disease (COPD), and other respiratory illnesses. This dissertational study investigated the effects of host factors in these viral infections. Chapter 2 and chapter 3 are related research about rhinovirus, and chapter 4 is research about influenza virus. Human rhinovirus (RV) is the major cause of common cold and it also plays a significant role in asthma and asthma exacerbation. Airway epithelium is the primary site of RV infection and production. In contrast, monocytic cells (e.g., monocytes and macrophages) are believed to be non-permissive for RV replication. Instead, RV has been shown to modulate inflammatory gene expressions in these cells via a replication-independent mechanism. However, Johnston et al. firstly reported a low-grade productive replication of RV9 in monocytes and also showed the RV16 replication in monocytes in following studies. In the present study, we generated the epithelial cell- monocyte coculture system. RV16 (a major-group RV) replication was found to be significantly enhanced in monocytes when co-cultivated with airway epithelial cells. This effect appeared to be mediated by secretory components from epithelial cells, which stimulated RV16 replication and significantly elevated the expression of a number of proinflammatory cytokines. The lack of such effect with RV1A, a minor-group RV that enters the cell by a different receptor, suggests that ICAM1, the receptor for major-group RVs, may be involved. Conditioned media from epithelial cells significantly increased ICAM1 expression in monocytes. Consistently, ICAM1 overexpression and ICAM1 knockdown enhanced and blocked RV production, confirming the role of ICAM1 in this process. In addition, we isolated secretory components from the epithelial conditioned medium and further determined their function. Alpha-Heremans Schmid Glycoprotein (AHSG) is the top matched secretory protein to induce the ICAM1 expression. High AHSG expression is closely related to asthma in mice models and human patients. Thus, we demonstrated that airway epithelial cells direct significant RV16 replication in monocytic cells via an ICAM1-dependent mechanism. This is the first time we demonstrated that AHSG expression is related to asthma. Influenza virus is the major cause of influenza (or the flu). Pandemic flu killed tens of millions of people and seasonal flu yearly outbreak also caused severe illness and hundreds of thousands of deaths. Current options for preventing or treating influenza are either limited (e.g., vaccine) or becoming ineffective due to the emergence of drug resistant strains (e.g., M2 blockers). Thus, new treatments for influenza viral infection are urgently needed. Host targets are relatively stable, so the drugs have high barrier when they are targeting on the host factors. To develop drugs targeting the host, it is important to understand the influenza related pathway in the host cells. In the present study, we generated an influenza infectious model on human airway epithelial cells, which are primary sites for the virus’ infections. We serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent and broad-spectrum activity against influenza. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza was insensitive to EGFR blockade by other EGFR inhibitors or by the knockdown using a small interference RNA against EGFR. Additionally, interferons are the major anti-viral proteins in the cells, its effect has to be considered when we research on the anti-viral activity. A knockout approach using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) further demonstrated that this antiviral activity was also interferon independent. AG1478 was found to target on Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1- ADP ribosylation factor 1 (GBF1-ARF1). AG1478 reversibly inhibit GBF1 activity and disrupted its Golgi-cytoplasmic trafficking. Compared to the two existing GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure, suggesting its antiviral activity may not originate from a non-specific disruption of intracellular vesicle trafficking regulated by GBF1. GBF1 was found to interact with a specific set of viral proteins including M1, NP and PA. AG1478-elicited spatial alternation of GBF1 distribution disrupted these interactions. Because host factors are more genetically stable than viral proteins, host-targeting antivirals might have a higher genetic barrier to drug resistance than direct-acting antivirals. In conclusion, the host factors are crucial for these respiratory viral illnesses, and drugs targeting host factors are a new direction for treating infectious illnesses. 1) For rhinovirus, we demonstrate that epithelial secretions, especially AHSG, direct robust RV replication in monocytes via significantly increased ICAM1. This new information will advance our understanding of the interaction between airway epithelium and inflammatory cells in the context of RV infection and RV-induced disease exacerbation. Blocking ICAM1 or decreasing AHSG expression will be a new direction in treating rhinovirus infections. This finding will open a new venue for the study of RV infection in airway disease and its exacerbation. 2) For influenza virus, through a serendipitous finding, we have discovered a potent and broad-spectrum anti-influenza drug candidate-AG1478. Its antiviral activity is mediated by targeting GBF1.The treatment of AG1478 disrupted this interaction and potentially impaired vRNP transport leading to markedly decreased IAV production. It is a new direction to develop the drugs targeting GBF1. Further development on this candidate target will lead to novel anti-influenza therapy.
Degree ProgramGraduate College
Pharmacology & Toxicology