USING SINGLE VIRION FUSION ASSAY TO STUDY INFLUENZA VIRUS ENTRY
Understanding the mechanism of influenza virus entry is critical for effectively developing anti-viral drugs and vaccines. The entry of influenza virus is mediated by two proteins: hemagglutinin (HA) and neuraminidase (NA). During virus infection, HA first binds to sialic acid moieties on epithelial cell membranes, triggering engulfment of the virus into the cell via endocytosis. As the endosome matures during trafficking, the pH decreases in the micro-environment, which triggers the conformational change of HA that induces membrane fusion of the viral and endosomal membranes. Fusion allows the virus to release viral RNA into the cytosol of the cell. Single particle tracking (SPT) enables the study of viral binding and membrane fusion at the single-virion level to obtain high resolution measurements of each of these processes in vitro. SPT combines total internal reflection fluorescence (TIRF) microscopy with microfluidics and supported lipid bilayers, making it a very powerful tool for host-pathogen membrane fusion studies. One concern about influenza is that the virus can mutate quickly via three mechanisms (i.e. error-prone RNA replication, influenza reassortment, and influenza recombination). Influenza virus mutation mechanisms may cause the emergence of new strains, some of which may lead to serious flu pandemics in humans. Understanding the fusion fitness of influenza strains at the single-virion level is crucial for the development of anti-viral fusion drugs which block genome transfer. However, high risk live influenza pandemic strains can be infectious and lethal to humans, which makes them a challenge to study in the laboratory environment without significant safety protocols in place. To study the influenza pandemic virus, using a pseudovirus is the most common surrogate system for studying virus entry. However, before this work, no studies directly compared the entry processes of pseudoviruses with their wildtype particles. Here I use SPT to compare the fusion kinetics of influenza native virus and its pseudovirus analog and show that these particles do recapitulate the native behavior well. I will then show how I use pseudoviruses to mimic influenza reassortment and shed light on how influenza reassortment can infect virus entry. Next, I report results on SPT studies of virus entry of influenza virus H10N8 (JX346), a virus of concern because, although there have been only 3 confirmed cases, it was transmitted directly from chicken to human in 2014, with high lethality. Lastly, I will expand the SPT technique to study the inhibitory mechanism of an influenza antiviral drug, IFITM3. IFITM3 has been shown to be able to stop influenza virus entry at the fusion step, and lead to the failure of infection.
Chemical engineering; Single particle tracking; influenza reassortment; Virology; single virion tracking; virus entry; influenza virus; Biophysics
Whittaker, Gary R.; Alabi, Christopher Akinleye
Ph. D., Chemical Engineering
Doctor of Philosophy
dissertation or thesis