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Viral Replication: Post-Integration Events
Monday, 1:30 - 3:30 pm
Background: HIV-1 particles assemble and bud selectively through lipid rafts in the plasma membrane of cells. Although the composition of the virus is similar to lipid rafts, contaminating cellular vesicles that co-purify with virions hinder attempts to characterize virion membrane organization. We have developed a new method, based on cholesterol addition to membranes, that allows for a simple, rapid, high yield separation of virions from contaminating vesicles by ultracentrifugation. We used these purified virus preparations to show that virions contain lipid rafts. We examined the effect of cholesterol loading on infectivity, morphology and distribution of gp120 in HIV and SIV. Finally, we show that permeabilization of virus, is correlated with a loss of lipid raft markers and raft-associated gp120.
Methods: Cholesterol loading is achieved by incubation of virus in an excess of cholesterol in the presence of 2-hydroxy-propyl-beta-cyclodextrin at 37oC. Infectivity was determined using a luciferase reporter cell line. Morphology was assessed by electron microscopic analysis. Virion-associated lipid rafts were isolated by incubation with 1% Triton X-100 on ice for 1 hour, and equilibrium ultracentrifugation on a 40%/38%/5% sucrose gradient. Immunodot analysis and Western blotting were used to assess membrane protein distribution in lipid raft preparations. Cholesterol was measured using a commercial kit (Molecular Probes).
Results: Cholesterol loading of virion preparations resulted in a significant reduction of CD45, a marker for cellular vesicle contamination, and a lack of recovery of vesicles in purified cellular vesicle preparations. Prominent changes in virion morphology followed superloading, however, titer was reduced by less than one log. In both HIV and SIV, gpi-anchored proteins and a fraction of antigen presentation molecules (MHC I and MHC II), and gp120 in SIV distributed to lipid raft regions, and were lost after permeabilization of virus.
Conclusions: Cholesterol loading is a rapid, efficient technique to purify virions. We show that virions contain lipid rafts. Since a fraction of gp120, and antigen presenting molecules are associated with lipid raft regions in virions, and that virions contain constitutively active integrins, we suggest that virions, by utilizing lipid rafts, may have the ability to form a type of modified immunological synapse with lymphocytes that may have profound consequences on viral pathogenesis.
Keywords: Lipid Rafts; Cholesterol; Structure