Rotavirus RRV associates with lipid membrane microdomains during cell entry

Rotavirus cell entry is a multistep process, not completely understood, which requires at least four interactions between the virus and cell surface molecules. In this work, we investigated the role of the sphingolipid- and cholesterol-enriched lipid microdomains (rafts) in the entry of rotavirus strain RRV to MA104 cells. We found that ganglioside GM1, integrin subunits alpha2 and beta3, and the heat shock cognate protein 70 (hsc70), all of which have been implicated as rotavirus receptors, are associated with TX-100 and Lubrol WX detergent-resistant membranes (DRMs). Integrin subunits alpha2 and beta3 were found to be particularly enriched in DRMs resistant to lysis by Lubrol WX. When purified RRV particles were incubated with cells at 4 degrees C, about 10% of the total infectious virus was found associated with DRMs, and the DRM-associated virus increased to 37% in Lubrol-resistant membrane domains after 60-min incubation at 37 degrees C. The virus was excluded from DRMs if the cells were treated with methyl-beta-cyclodextrin (MbetaCD). Immunoblot analysis of the viral proteins showed that the virus surface proteins became enriched in DRMs upon incubation at 37 degrees C, being almost exclusively localized in Lubrol-resistant DRMs after 60 min. These data suggest that detergent-resistant membrane domains play an important role in the cell entry of rotaviruses, which could provide a platform to facilitate the efficient interaction of the rotavirus receptors with the virus particle.