To Study the Multiple Roles of Ddx3 in Viral/Bacterial Infections and Inflammation

Human DDX3 is a member of DEAD-box RNA helicases that play important roles in eukaryotic gene expression. DDX3 and its homologs have been implicated in many aspects of mRNA metabolism, including translation. Previously, we found that DDX3 is recruited to the cytoplasmic stress granules (SGs) under cell stress conditions, suggesting a role for DDX3 in translational control. DDX3 is not required for general translation, but it can facilitate translation of selective mRNAs that contain a long or structured 5’ untranslated region (UTR). DDX3 may facilitate ribosome scanning by resolving secondary structures in the 5’ UTR of selective mRNAs during translation initiation. It has recently emerged that DDX3 plays a role in antiviral innate immunity. However, the precise role of DDX3 in antiviral innate immunity is still not fully understood. We recently demonstrated that DDX3 is required for translation of the protein activator of the interferon-induced protein kinase (PACT). PACT is a double-stranded RNA (dsRNA)-binding protein (dsRBP) that functions as a cellular activator of RIG-I to facilitate viral RNA recognition. In this project, we will provide compelling evidence that DDX3 functions in bacterial infection and inflammation for the first time. We found that DDX3 is required for translation of Stat1, Gnb2, Rac1, TAK1 and p38 MAPK mRNAs in THP-1 cells and HeLa cells. PACT, Stat1, Gnb2, Rac1, TAK1 and p38 MAPK proteins play different roles in the regulation of innate immunity. Therefore, we propose that DDX3 may play multiple roles in viral/bacterial infections and inflammation. To test this hypothesis, three specific aims are designed in this research project: Specific aim 1 will study translational control of DDX3 in viral/bacterial infections and inflammation. Specific aim 2 will determine whether DDX3 participates in macrophage migration and phagocytosis. Specific aim 3 will elucidate the importance of DDX3 in the inflammatory response during viral/bacterial infections. To accomplish the above-mentioned research objectives, standard procedures including plasmid constructs, cell culture and transfection, lentivirus-mediated RNAi knockdown, translation assay, site-directed mutagenesis, immunoblotting, in vitro transwell cell migration assay, in vitro macrophage phagocytosis assay, assay, colony formation assay, flow cytometry analysis, human cytokine array, enzyme-linked immunosorbent assay (ELISA), quantitative real-time RT-PCR will be conducted. We expect to finish this proposed project within one year.

Project ID:PC10708-1126
External Project ID:MOST107-2320-B182-031