RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors

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RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors

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dc.contributor.advisor Hacohen, Nir
dc.contributor.author DeGrace, Marciela
dc.date.accessioned 2013-02-21T22:51:03Z
dc.date.issued 2013-02-21
dc.date.submitted 2012
dc.identifier.citation DeGrace, Marciela. 2012. RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors. Doctoral dissertation, Harvard University. en_US
dc.identifier.other http://dissertations.umi.com/gsas.harvard:10152 en
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:10330308
dc.description.abstract Influenza 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. en_US
dc.language.iso en_US en_US
dash.license META_ONLY
dc.subject Hermansky-Pudlak syndrome en_US
dc.subject HPS1 en_US
dc.subject influenza en_US
dc.subject RNAi en_US
dc.subject viral entry en_US
dc.subject virology en_US
dc.subject immunology en_US
dc.subject cellular biology en_US
dc.title RNAi Screens in Primary Human Lung Cells Reveal Hermansky-Pudlak Syndrome Proteins as Influenza Suppressors en_US
dc.type Thesis or Dissertation en_US
dash.embargo.until 10000-01-01
thesis.degree.date 2012 en_US
thesis.degree.discipline Virology en_US
thesis.degree.grantor Harvard University en_US
thesis.degree.level doctoral en_US
thesis.degree.name Ph.D. en_US
dc.contributor.committeeMember DeCaprio, James en_US
dc.contributor.committeeMember Gehrke, Lee en_US
dc.contributor.committeeMember Tager, Andrew en_US
dc.contributor.committeeMember Kagan, Jonathan en_US
dc.contributor.committeeMember Connor, John en_US
dc.contributor.committeeMember Farzan, Michael en_US

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TableS6.xls 420Kb Microsoft Excel View/Open
TableS5.xls 220Kb Microsoft Excel View/Open

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