Background: Recent evidence suggests HIV-1 particles assemble and bud selectively through areas in the plasma membrane of cells that are highly enriched with GPI-anchored proteins and cholesterol, called lipid rafts. Since cholesterol is required to maintain lipid raft structure and function, we hypothesized that compounds able to remove cholesterol from membranes might be disruptive to HIV-1 and SIV. We have previously shown that membrane-cholesterol removal from cell-free or cell-associated HIV-1 with the compound 2-hydroxy-propyl-beta-cyclodextrin (b-CD) renders both virus types non-infectious by preventing membrane fusion. Here we have further examined the effect of b-CD on the structure and infectivity of cell-free virions.

Methods: Viral infectivity was determined using a SIV luciferase tat transcomplementation reporter cell line, and virion integrity was examined by HPLC, Western blotting, and Electron Microscopic analysis. Virion-associated cholesterol was measured using an AMPLEX red cholesterol kit (Molecular Probes).

Results: We found that b-CD inactivated HIV-1 and SIV in a dose dependent manner and permeabilized the viral membrane, resulting in the loss of mature Gag proteins (capsid, matrix, nucleocapsid, p1, and p6), without loss of the envelope glycoprotein. Host proteins located internally in the virus (actin, moesin, and ezrin) and membrane-associated host proteins (HLA-A and HLA-DR) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viral membrane, holes were present in the virus membrane and viral core structure was perturbed. Host lipid raft markers incorporated into the virion membrane were lost upon permeabilization.

Conclusions: These data provide evidence that an intact viral membrane is required to maintain mature virion core integrity. Since host cytoskeletal proteins were not affected and virion particles were recovered, the data suggest that there may be an organized scaffold inside virion particles that can maintain overall structure despite disruptions in membrane integrity. The loss of lipid raft markers upon virus permeabilization and a biphasic pattern of cholesterol depletion from the viral membrane also provides strong evidence for the presence of organized membrane microdomains that are incorporated into the virion particle.