Previous reports have documented that
cholesterol supplementations increase cytopathic effects in tissue culture and also intensify in vivo pathogenicities during
infection by the enveloped coronavirus murine hepatitis virus (MHV). To move toward a mechanistic understanding of these phenomena, we used growth media enriched with
methyl-beta-cyclodextrin or
cholesterol to reduce or elevate cellular membrane
sterols, respectively.
Cholesterol depletions reduced plaque development 2- to 20-fold, depending on the infecting MHV strain, while supplementations increased susceptibility 2- to 10-fold. These various
cholesterol levels had no effect on the binding of viral spike (S)
proteins to cellular
carcinoembryonic antigen-related
cell adhesion molecule (CEACAM) receptors, rather they correlated directly with S-
protein-mediated membrane fusion activities. We considered whether
cholesterol was indirectly involved in membrane fusion by condensing CEACAMs into "
lipid raft" membrane microdomains, thereby creating opportunities for simultaneous binding of multiple S
proteins that subsequently cooperate in the receptor-triggered membrane fusion process. However, the vast majority of CEACAMs were solubilized by cold
Triton X-100 (TX-100), indicating their absence from
lipid rafts. Furthermore, engineered CEACAMs appended to
glycosylphosphatidylinositol anchors partitioned with TX-100-resistant
lipid rafts, but cells bearing these raft-associated CEACAMs were not hypersensitive to MHV
infection. These findings argued against the importance of
cholesterol-dependent CEACAM localizations into membrane microdomains for MHV entry, instead suggesting that
cholesterol had a more direct role. Indeed, we found that
cholesterol was required even for those rare S-mediated fusions taking place in the absence of CEACAMs. We conclude that
cholesterol is an essential membrane fusion cofactor that can act with or without CEACAMs to promote MHV entry.