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Identification of host factors in swine respiratory epithelial cells that contribute to host anti-viral defense and influenza virus replication



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Swine influenza viruses (SIV) are a common and an important cause of respiratory disease in pigs. Pigs can serve as mixing vessels for the evolution of reassortment viruses containing both avian and human signatures, which have the potential to cause pandemics. NS1 protein of influenza A viruses is a major antagonist of host defence and it regulates multiple functions during infection by interacting with a variety of host proteins. Therefore, it is important to study swine viruses and NS1-interacting host factors in order to understand the mechanisms by which NS1 regulates virus replication and exerts its host defense functions. Influenza A viruses enter the host through the respiratory tract and infect epithelial cells in the respiratory tract, which form the primary sites of virus replication in the host. Thus, studying SIV infection in primary swine respiratory epithelial cells (SRECs) would resemble conditions similar to natural infection. The objectives of this study were to identify NS1-interacting host factors in the virus-infected SRECs and to understand the physiological role of at least one of the factors in influenza virus infection. The approaches to meet this objective were to generate a recombinant SIV carrying a Strep-tag in the NS1 protein, infect SRECs with the Strep-tag virus, purify NS1-interacting host protein complex from the infected cells by pull-down using strep-tactin resin and then study the physiological role of one of the NS1-interacting partners during influenza infection. Using a reverse-genetics strategy, a recombinant virus carrying the Strep-tag NS1 was successfully rescued and the SRECs were infected with this recombinant virus. The Strep-tag in the NS1 protein facilitated the isolation of an intact NS1-interacting protein complex and the proteins present in the complex were identified by liquid chromatography-tandem mass spectrometry. The identified proteins were grouped to enrich for different functions using bioinformatics. This gave an insight into the different functions that NS1 may regulate during infection and the potential host partners involved in these functions. Among the host proteins identified as potential interaction partners, RNA helicases were particularly of interest to study. Influenza being an RNA virus, RNA helicases could have important functions in the virus life cycle. Among the identified RNA helicases, DDX3 has been shown to regulate IFNβ induction and affect the life cycle of a number of viruses. However, its function in influenza A virus life cycle has not been studied. Hence, this study explored whether DDX3 has any role in the influenza A virus life cycle. Immunoprecipitation studies revealed viral proteins NP and NS1 as direct interaction partners with DDX3. DDX3 is a known component of stress granules (SGs) and influenza A virus lacking the NS1 gene is reported to induce SG formation. Therefore, the role of DDX3 in SG formation, induced by PR8 influenza A virus lacking NS1 (PR8 del NS1) was explored. The results from this study showed that DDX3 co-localized with NP in SGs indicating that DDX3 may interact with NP in the SGs. NS1 protein was found to inhibit virus-induced SGs and DDX3 downregulation impaired virus-induced SG formation. The contribution of the different domains of DDX3 to viral protein interaction and virus-induced SG formation was also studied. While DDX3 helicase domain did not interact with NS1 and NP, it was essential for DDX3 localization in virus induced SGs. Moreover, DDX3 downregulation resulted in the increased replication of PR8 del NS1virus, accompanied by an impairment of SG induction in infected cells. Since DDX3 is reported to regulate IFNβ induction, the role of DDX3 in influenza A virus induced IFNβ induction was also examined. Using small molecule inhibitors and siRNA-mediated gene knockdown, the RIG-I pathway was identified as the major contributor of influenza induced IFNβ induction in newborn porcine tracheal epithelial (NPTr) cells. DDX3 downregulation and overexpression also showed that DDX3 has an inhibitory effect on IFNβ expression induced by both influenza infection and low molecular weight (LMW) poly I:C treatment, which is also a RIG-I ligand. RNA competition assay to identify the mechanism of DDX3-mediated inhibition, showed that RIG-I binding affinity to its ligands LMW poly I:C and influenza viral RNA (vRNA) is much higher than that of DDX3. Furthermore, DDX3 downregulation enhanced titers of the PR8 del NS1 virus, while it did not affect the titers of the wild-type strains of PR8 and SIV/SK viruses. Overall, the results show that DDX3 has an antiviral role and the SG regulatory function of DDX3 has a profound effect on virus replication than the IFNβ regulatory function.



Influenza, NS1, Strep-tag, DDX3, Stress granules, Innate immunity, IFN beta



Doctor of Philosophy (Ph.D.)


School of Public Health


Vaccinology and Immunotherapeutics


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