Application of New Tools for Probing the Roles of Sphingolipids and Cholesterol in Influenza Virus Infection

项目总结

PROJECT SUMMARY Influenza A virus (IAV) is a major public health threat that causes 290,000 to 650,000 deaths per year worldwide, while pandemic strains have caused millions of deaths. Despite the existence of antiviral drugs and vaccines, IAV still causes high mortality and morbidity due to its ability to rapidly mutate and escape herd immunity. Identification of the host cell components that promote infection when present at high levels but sustain normal cell function at lower levels could lead to the development of new types of antiviral therapeutics with universal strain potency and decreased drug resistance. Sphingolipids and cholesterol are potential targets because IAV replication and infectivity is correlated with their abundances in the host cell. Why high levels of sphingolipids and cholesterol seem to promote IAV infection remains a mystery. Likewise, which steps in the virus lifecycle require these membrane components have not been identified. The research proposed herein will use new modeling and experimental strategies to investigate the roles of sphingolipids and cholesterol in the IAV lifecycle. These cutting-edge strategies include molecular dynamic (MD) simulations for modeling IAV fusion peptide (HAfp) insertion into membranes, and a high-resolution secondary ion mass spectrometry (SIMS) technique for imaging cholesterol and sphingolipids on and within cells. Three aims that use these techniques are proposed. In Aim 1 the molecular interactions between different lipid species, cholesterol, and the HAfp will be characterized by using MD simulations to determine how these cholesterol and sphingolipids affect HAfp insertion into the endosomal membrane. Completion of this aim will provide understanding of how the HAfp interacts with distinct membrane lipids, which is a necessary perquisite for elucidating the roles of membrane composition in IAV fusion during entry. In Aim 2, the sphingolipid and cholesterol abundances at the sites where the IAV assembles and buds from the host cell plasma membrane will be identified by using a combination of metabolic rare stable isotope incorporation, immunolabeling, and high-resolution SIMS imaging. This will be a decisive test of the hypothesis that the influenza A virus assembles and buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. In Aim 3, high-resolution SIMS depth profiling, an innovative 3D image reconstruction tool developed by sponsor’s lab, and statistical hypothesis tests will be used to visualize and compare the relative abundances of cholesterol in compartments that contain the influenza virus envelope protein, hemagglutinin (HA), within IAV-infected and uninfected cells. These studies will test the hypothesis that cholesterol levels are elevated in the compartments involved in trafficking newly synthesized IAV proteins to the plasma membrane for assembly into progeny virus particles. Successful completion of these aims may identify key host cell factors that might be exploited to combat IAV infection.