RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors
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CitationDeGrace, Marciela. 2012. RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors. Doctoral dissertation, Harvard University.
AbstractInfluenza is an important human pathogen that causes fatal disease in 250,000-500,000 people worldwide each year. Because of high levels of variation between influenza strains, vaccines are not always effective and must be administered annually. Influenza virus, which replicates primarily in the lung epithelium, encodes only 10 proteins and relies heavily on host products to replicate. Determining which cellular factors are important for influenza replication represents an important area of virology and cellular biology research, and could elucidate proteins or pathways to target for antiviral therapies. We developed a high throughput screening method in primary human bronchial epithelium (HBECs) to identify novel regulators of influenza replication. We first used this method to functionally examine 1745 genes that were identified as potential influenza regulators due to transcriptional regulation by virus or viral products, direct interaction with viral proteins via yeast two-hybrid, or through computational analysis. This screen confirmed some known regulators of influenza replication while identifying novel viral interactors as influenza regulators (e.g. USHBP1, ZMAT4). We also found that the WNT, p53, and ER stress pathways, among others, affect viral replication and interferon production. The life cycle of influenza involves extensive intracellular trafficking of viral components. We again used RNAi to systematically examine the roles of vesicle, RNA, and protein trafficking genes in the production of infectious influenza A virus in primary lung cells. Among the factors that significantly impact viral infection, we identify a set of five genes with strong antiviral effects that are mutated in patients with Hermansky-Pudlak syndrome (HPS). Depletion of HPS genes leads to elevated viral RNA at an early stage of influenza infection prior to transcription. In contrast, depletion of these genes does not alter the innate immune response to virus or interferon. Using an HPS-1 patient cell line, we find an increase in viral fusion to endosomal compartments but no change in viral binding to the cell surface or entry into the early endosome. Our studies uncover a potential role for many trafficking factors in the influenza life cycle, and point to an HPS1-dependent process that inhibits viral entry prior to viral membrane fusion.
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