| Lipid rafts are involved in several important cellular processes including; endocytosis, vesicle transport, cholesterol sorting, apoptosis and signaling through the T cell receptor. We have proposed that HIV and SIV may incorporate a lipid raft into the virion particle. Biochemical characterization of virions is limited by the co-purification of vesicles of the same density as virions, exosomes and plasma membrane fragments, collectively termed microvesicles. Here we develop a new method based upon cholesterol loading of virus preparations to generate highly purified virion preparations, which show a marked loss of CD45, a marker for cellular contaminants, and microvesicles by EM. Using these purified virus preparations, we confirm the presence of virion-associated lipid rafts in HIV and SIV, and that a fraction of gp120 is associated with rafts in SIV. Cholesterol loaded virions demonstrate an altered morphology, which includes the presence of a prominent membrane bleb, but minimal differences in infectivity. Virion associated lipid rafts organize a proportion of MHC I and MHC II, and other molecules involved in the formation of the T-cell synapse are arranged in an analogous manner to how they are organized in cells. Since cholesterol is required to maintain lipid raft structure and function, we proposed that cholesterol removal with the compound 2-hydroxy-propyl-beta-cyclodextrin (beta-CD) might be disruptive to HIV-1 and SIV. We therefore examined the effect of beta-CD on the structure and infectivity of cell-free virions. beta-CD inactivated HIV-1 and SIV in a dose dependent manner and permeabilized the viral membranes, resulting in the loss of mature Gag proteins, without loss of the envelope glycoproteins. Host proteins located internally in the virus (actin, moesin and ezrin) and membrane associated host proteins (MHC class I and II) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viruses, holes were present in the viral membranes and viral core structure was perturbed. The loss of membrane is correlated with a loss of lipid raft markers, and we suggested that this missing section of membrane is a virion-associated lipid raft. The lost lipid raft markers could be recovered from the supernatant by co-immunoprecipitation, and ultracentrifugation, and were in a complex that could be disrupted by detergent treatment, suggesting that the lost segment of viral membrane is stable and may be organized into a lipid raft-like structure. We found that lipid raft markers, and a small subset of gp120 and matrix protein were recovered in a large molecular weight complex at 48S. Collectively, these data provide direct evidence for a highly ordered, stable microdomain or virion-associated lipid raft that is incorporated into lentiviral particles, we term the Lentiviral integral disk (LID) and a protein scaffold that can maintain overall structure despite disruptions in membrane integrity we term the viroskeleton. |
