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. The formation of infectious influenza virions and the clustering of influenza virus proteins in the plasma membrane during virus assembly are reportedly reduced by depletion of cellular cholesterol. Though this cholesterol sensitivity certainly suggests that cholesterol plays a role in influenza virus infection, whether the plasma membrane domain from which the influenza virus buds is enriched with cholesterol and sphingolipids has not been decisively determined. We have addressed this question by using high-resolution secondary ion mass spectrometry (SIMS) to image the distributions of stable isotope-labeled lipids and immunolabeled influenza virus proteins in the plasma membranes of virus-infected cells with ∼100 nm lateral resolution. In these experiments, we metabolically incorporated the distinct stable isotopes 15N and 18O into the sphingolipids and cholesterol, respectively, in living MDCK cells. We infected these MDCK cells with an H3N2 strain of the influenza virus for 24 h, and immunolabeled the influenza envelope protein, hemagglutinin, to permit the detection of budding virus. Finally, we imaged the metabolically incorporated 15N-sphingolipids and 18O-cholesterol and immunolabeled influenza hemagglutinin on the surfaces of the influenza-infected MDCK cells, and assessed co-localization between these components. The results of these experiments and there implications will be presented.