Cholesterol is important for a post-adsorption step in the entry process of transmissible gastroenteritis virus.

Cholesterol is a major constituent of detergent-resistant membrane microdomains (DRMs). We localized transmissible gastroenteritis virus (TGEV) spike (S) protein in DRMs in the viral envelope. Though S protein was not solubilized by cold non-ionic detergents, this behavior was unchanged when cholesterol was depleted from viral membrane by methyl-ß-cyclodextrin (MßCD) and the protein did not comigrate with cellular DRM marker proteins in flotation analyses. Therefore, the S protein is not anchored in the viral membrane DRMs as they are known to occur in the plasma membrane. Cholesterol depletion from viral membrane may not affect the adsorption process as neither the sialic acid binding activity nor the binding to aminopeptidase N was reduced post-MßCD treatment. Reduced infectivity of cholesterol-depleted TGEV was observed only when the adsorption process occurred at 37°C but not when the virus was applied at 4°C. Cholesterol is important for a post-adsorption step, allowing membrane rearrangements that facilitate virus entry.

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.

Importance of cholesterol for infection of cells by transmissible gastroenteritis virus.

In this study, we addressed the question whether cholesterol is important for transmissible gastroenteritis virus (TGEV), a porcine coronavirus, in the initiation of an infection. We found that cholesterol depletion from the cellular membrane by methyl-beta-cyclodextrin (MbetaCD) significantly impaired the efficiency of TGEV infection. Infectivity was also reduced after depleting cholesterol from the viral envelope. This finding is surprising because coronaviruses bud from a pre-Golgi compartment which is expected to be low in cholesterol compared to the plasma membrane. Addition of exogenous cholesterol resulted in a restoration of the infectivity confirming our conclusion that efficient TGEV infection requires cholesterol in both the viral and the cellular membranes. Our data raise the possibility that the viral and cellular proteins involved in the entry process may be associated with cholesterol-rich membrane microdomains.