Does the Influenza Virus Bud from Cholesterol- and Sphingolipid-Enriched Plasma membrane domains?

Abstract

The segregation of different lipid and protein species into compositionally distinct membrane domains is undoubtedly essential for cellular function. Advances in protein imaging have vastly improved our knowledge of the distributions and stimuli-dependent clustering of various protein species within cellular membranes. However, much less is known about the distributions of distinct lipid species, and especially their proximity to proteins of interest, within the plasma membrane. We have shown that high-resolution secondary ion mass spectrometry (SIMS) performed on a Cameca NanoSIMS 50 enables imaging the distributions of stable isotope-labeled lipids in model and cellular membranes with ∼100 nm lateral resolution. Our previous NanoSIMS imaging of metabolically incorporated 15N-sphingolipids and 18O-cholesterol on the surfaces of fibroblast cells revealed the plasma membrane contains cytoskeleton-dependent sphingolipid domains that are not enriched with cholesterol. Unexpectedly, cholesterol was found to be uniformly distributed within the plasma membrane. Here we use this approach to test the long-standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. Specifically, we have imaged metabolically incorporated 15N-sphingolipids and 18O-cholesterol and immunolabeled viral envelope proteins on the surfaces of influenza-infected MDCK cells, and assessed co-localization between these components. The results of these experiments and there implications will be presented.