Functional Evaluation Of Flag-Stat1 In U3a Cells
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Date
2017
Authors
Tey, Lee Hung
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Viral infections have had massive socio-economic impact, at the costs of human health and invaluable lives. However, effective and broad spectrum protection against viruses is still lacking despite best efforts. Antiviral immune defense is crucial for host protection in the event of viral infections. Type I interferons (IFN), are key mediators for antiviral immunity, and are induced following recognition of viral-associated molecular patterns. Type I IFNs released to the extracellular matrix then help establish antiviral state at the cellular level in autocrine and paracrine manner. Signaling by Type I IFNs is transduced via Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, leading to upregulation of interferon stimulated genes. STAT1 is a key component of the JAK-STAT signaling pathway, where two major isoforms: STAT1A and STAT1B are naturally expressed during Type I IFN response. STAT1 is essential in antiviral immunity as Stat1-/- mice are more susceptible to virus infection. However, the possibly of distinct roles of STAT1 isoforms in antiviral immunity warrants further investigation for more comprehensive understanding of the JAK-STAT pathway towards developing protection against viruses. This project thereby serves as groundwork in preparation for future research on STAT1 isoforms and their cellular functions. We aim to establish an essay
involving the expression of a functional recombinant Flag-STAT1B in U3A cells, a cell line lacking endogenous STAT1 that is derived from 2fTGH cells. Therefore, expression plasmid for recombinant Flag-STAT1B was constructed by sub-cloning. The expression plasmid was transfected into U3A cells and Flag-STAT1B was successfully expressed. When the transfected cells were challenged with IFN-β, recombinant Flag-STAT1B partially restored the previously dysfunctional JAK-STAT signaling pathway in U3A cells. Protein expression levels of several interferon stimulated genes were semi-quantitatively analyzed through immunoblotting and ImageJ. From our results, overexpression of STAT1B without STAT1A in U3A cells could not fully restore the JAK-STAT signaling pathway. While more research is necessary, our work suggest that the different STAT1 isoforms may have overlapping but yet distinct roles at the cellular level during Type I interferon response. Further investigation may lead to novel understanding about the mechanism of STAT1 isoforms and their impact on host antiviral immunity.
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Keywords
Functional evaluation of , flag-stat1 in u3a cells