Importance of cholesterol-rich membrane microdomains in the interaction of the S protein of SARS-coronavirus with the cellular receptor angiotensin-converting enzyme 2

Cholesterol present in the plasma membrane of target cells has been shown to be important for the Infection by SARS-CoV. We show that Cholesterol depletion by treatment with methyl-beta-cyclodextrin (m beta CD) affects Infection by SARS-CoV to the same extent as Infection by vesicular stomatitis virus-based pseudotypes containing the surface glycoprotein S of SARS-CoV (VSV-Delta G-S). Therefore, the role of Cholesterol for SARS-CoV Infection can be assigned to the S protein and is unaffected by other coronavirus proteins. There have been contradictory reports whether or not angiotensin-converting Enzyme 2 (ACE2), the cellular receptor for SARS-CoV, is present in detergent-resistant membrane domains. We found that ACE2 of both Vero E6 and Caco-2 cells co-purifies with marker proteins of detergent-resistant membranes supporting the notion that cholesterol-rich microdomains provide a platform facilitating the efficient interaction of the S protein with the cellular receptor ACE2. To understand the involvement of Cholesterol in the initial steps of the viral life cycle, we applied a cell-based binding assay with cells expressing the S protein and cells containing angiotensin-converting Enzyme 2 (ACE2). Alternatively, we used a soluble S protein as interaction partner. Depletion of Cholesterol from the ACE2-expressing cells reduced the binding of S-expressing cells by 50% whereas the binding of soluble S protein was not affected. This result suggests that optimal Infection requires a multivalent interaction between viral attachment protein and cellular receptors.