Lipid rafts, exosomes, and human T-cell leukemia virus type 1 biology: A new model of viral pathogenesis

Previous studies in the Hildreth laboratory determined that the expression of VCAM-1 in the erythroleukemic cell line K562 caused susceptibility to HTL-V-1-induced syncytium formation. The observation that not all cells expressing VCAM-1 are sensitive to syncytium formation by the virus indicated that K562 cells express a second molecule which, in the presence of VCAM-1, renders the cells susceptible to syncytium formation. Immunization of mice with K562 cells and generation of hybridomas resulted in four monoclonal antibodies that blocked HTLV-1-induced syncytium formation between K562/VCAM1 cells and HTLV-1-infected MT2 cells. Cell phenotyping and immunoblotting indicated that all four MAbs recognized distinct proteins. Treatment of cells with phosphatidylinositol-specific phospholipase C determined that three of the four MAbs recognized GPI-anchored proteins, which are enriched in lipid raft regions of the plasma membrane. Lipid raft isolation and immunoblot analysis determined that proteins recognized by all four MAbs localized largely if not entirely to lipid rafts. Dispersion of lipid rafts by cholesterol depletion inhibited HTLV-1-induced syncytium formation without affecting the ability of VCAM-1 to bind its ligand, indicating the importance of lipid rafts in HTLV-1 biology. One of the four MAbs, K5.M1, whose target protein was deemed a particularly strong candidate as a virus receptor, recognized a 17 kD protein. Through affinity purification, this protein was isolated, sequenced by tandem mass spectrometry, and identified as Leu-13 antigen. Lipid raft marker CD59 was found on the viral envelope as determined by virus capture assays, and we investigated whether HTLV-1 used lipid rafts as sites for budding. Immunoblot, virus capture, and confocal microscopy data indicated that proteins on HTLV-1 virions are found on lipid raft domains in infected cells. However, some proteins which were in lipid rafts were not found on the virus. This observation, in addition to the high levels of MHC class II and tetraspanner protein incorporation into the virions led us to examine the possibility of a subcellular origin of the virions. The data presented in this study indicates that HTLV-1 virions carry many traits associated with exosomes.