Person: Sun, Eileen
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Eileen
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Sun, Eileen
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Publication Dual Function of CD81 in Influenza Virus Uncoating and Budding(Public Library of Science, 2013) He, Jiang; Sun, Eileen; Bujny, Miriam V.; Kim, Doory; Davidson, Michael W.; Zhuang, XiaoweiAs an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.Publication Investigating Host Protein Function and Developing Assays for Influenza Virus Infection(2015-04-08) Sun, Eileen; Hogle, James; Cunningham, James; Nibert, Max; Shaw, MeganWith eight genomic segments encoding at least thirteen proteins, influenza virus can subvert a cell into a virus-producing factory. To study influenza infection, I utilize versatile fluorescent-based technologies to non-invasively probe a range of biological processes and targets in both static and living systems. This thesis covers three broad areas aimed towards unraveling the complexities of influenza infection: understanding how host proteins regulate viral infection, developing assays to study infection heterogeneity, and applying live cell imaging to study antiviral mechanism of action. First, I discuss how two cellular proteins—COPI complex and CD81—facilitate influenza infection. Genome-wide knockdown screens identified COPI complex and CD81 as influenza host dependency factors, but their specific function remained unclear. Applying imaging and flow cytometry methods, I found COPI siRNA knockdown inhibited virus entry during internalization and transport to late endosomes, and late stage infection during viral membrane protein trafficking. However, acute pharmacological treatment only recapitulated membrane protein trafficking defects, suggesting COPI directly facilitates late stage infection, not entry. In contrast, CD81 facilitates both viral fusion during entry and scission during egress. Single particle tracking studies revealed ~50% of virus particle fusion events occurred within CD81+ endosomes, and CD81 is recruited to progeny virus budding zones to facilitate viral scission. Second, while influenza infection encompasses a complex mixture of incomplete infection events, no influenza infection assay has thus far comprehensively evaluated infection heterogeneity. To study the prevalence and composition of incomplete infection events, I developed a multiplexed immunofluorescence assay to probe viral protein expression from all eight genomic segments. I found that influenza infection heterogeneity is highly prevalent, and the composition of different infection states exhibits correlated expression patterns amongst a subset of viral proteins. Lastly, I include a study applying live cell imaging to dissect the mechanism of a newly identified influenza antiviral drug, clotrimazole. I found clotrimazole inhibited WSN influenza infection between viral fusion and replication. Altogether, a multi-pronged approach is required to study the complex influenza infection cycle. Deciphering the complexities will guide development of much needed influenza therapeutics and vaccines.