Is the site of influenza virus assembly and budding enriched with cholesterol and sphingolipids?

Abstract

The observation that the influenza viral envelope is enriched with cholesterol and sphingolipids contributed to the hypothesis that the influenza virus assembles and bud from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane. Protein imaging experiments have demonstrated that the clustering of influenza virus proteins in the plasma membrane is sensitive to depletion of cellular cholesterol. However, the precise distributions of cholesterol and sphingolipids in relation to the site of influenza budding remains speculative due to an inability to directly image lipids without the use of potentially perturbing labels. We have used high‐resolution secondary ion mass spectrometry (SIMS), which is performed on a Cameca NanoSIMS 50, to image the distributions of stable isotope‐labeled lipids in the plasma membranes of intact cells with ~100 nm lateral resolution. Here we report using this approach to directly test the long‐standing hypothesis that the influenza virus buds from plasma membrane domains that are enriched with cholesterol and sphingolipids. We imaged metabolically incorporated 15N‐sphingolipids and 18O‐cholesterol and immunolabeled influenza virus envelope proteins on the surfaces of influenza‐infected MDCK cells with high‐resolution SIMS. The sites of virus assembly and budding in the plasma membrane were identified as the regions on the MDCK cell that were enriched with the immunolabeled influenza virus envelope protein, hemagglutinin. High‐resolution SIMS imaging revealed the sites of influenza virus assembly and budding were not enriched with cholesterol and sphingolipids. Although sphingolipid domains were detected in the plasma membranes of these MDCK cells, they had little co‐localization with the hemagglutinin‐rich membrane patches. These findings argue against the hypothesis that the influenza virus buds from cholesterol‐ and sphingolipid‐enriched domains in the plasma membrane.Support or Funding InformationThis research was supported by the U.S. National Science Foundation under CHE 15‐08662.