The lipid raft-associated protein CD98 is required for vaccinia virus endocytosis

Mature vaccinia virus (vaccinia MV) infects a broad range of animals in vivo and cell cultures in vitro; however, the cellular receptors that determine vaccinia MV tropism and entry pathways are poorly characterized. Here, we performed quantitative proteomic analyses of lipid raft-associated proteins upon vaccinia MV entry into HeLa cells. We found that a type II membrane glycoprotein, CD98, is enriched in lipid rafts upon vaccinia MV infection compared to mock-infected HeLa cells. The knockdown of CD98 expression in HeLa cells significantly reduced vaccinia MV entry. Furthermore, CD98 knockout (KO) mouse embryonic fibroblasts (MEFs) also exhibited reduced vaccinia MV infectivity without affecting MV attachment to cells, suggesting a role for CD98 in the postbinding step of virus entry. Further characterization with inhibitors and dominant negative proteins that block different endocytic pathways revealed that vaccinia MV entry into MEFs occurs through a clathrin-independent, caveolin-independent, dynamin-dependent, fluid-phase endocytic pathway, implying that CD98 plays a specific role in the vaccinia MV endocytic pathway. Infections of wild-type and CD98 KO MEF cells with different strains of vaccinia MV provided further evidence that CD98 plays a specific role in MV endocytosis but not in plasma membrane fusion. Finally, different CD98-C69 chimeric proteins were expressed in CD98 KO MEFs, but none were able to reconstitute MV infectivity, suggesting that the overall structure of the CD98 protein is required for vaccinia MV endocytosis.