Investigating Host Protein Function and Developing Assays for Influenza Virus Infection

Abstract

With 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.