c-Cbl-mediated selective virus-receptor translocations into lipid rafts regulate productive Kaposi's sarcoma-associated herpesvirus infection in endothelial cells.

During target cell entry and infection, many enveloped and nonenveloped viruses utilize cell surface receptors that translocate into lipid rafts (LRs). However, the mechanism behind this translocation is not known. Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with the human microvascular dermal endothelial (HMVEC-d) cell surface heparan sulfate (HS), integrins α3ß1, αVß3, and αVß5, and the amino acid transporter x-CT protein and enters via c-Cbl-bleb-mediated macropinocytosis (Veettil et al., J. Virol. 82:12126-12144, 2008; Veettil et al., PLoS Pathog. 6:e1001238, 2010). Here we have demonstrated that very early during infection (1 min postinfection), c-Cbl induced the selective translocation of KSHV into the LR along with the α3ß1, αVß3, and x-CT receptors but not αVß5. Activated c-Cbl localized with LRs at the junctional base of macropinocytic blebs. LR-translocated α3ß1 and αVß3 were monoubiquitinated, leading to productive macropinocytic entry, whereas non-LR-associated αVß5 was polyubiquitinated, leading to clathrin entry that was targeted to lysosomes. c-Cbl knockdown blocked the macropinocytosis and receptor translocation and diverted KSHV to a clathrin-lysosomal noninfectious pathway. Similar results were also seen by LR disruption with MßCD. These studies provide the first evidence that c-Cbl regulates selective KSHV-α3ß1, -αVß3, and -x-CT receptor translocations into the LRs and differential ubiquitination of receptors which are critical determinants of the macropinocytic entry route and productive infection of KSHV. Our studies suggest that interventions targeting c-Cbl and LRs are potential avenues to block KSHV infection of endothelial cells.