10th Conference on Retroviruses and Opportunistic Infections Boston, MA USA - February 10 -14, 2003

Conf Retroviruses Opportunistic Infect 2003 Feb 10-14;10th: abstract no. 18
M. Cavrois¹ , W. Yonemoto¹, D. Fenard¹, J. Neidleman ¹, W. Greene^1,2
1Gladstone Inst of Virology and Immunology, San Francisco, CA and ²Univ of California at San Francisco

BACKGROUND: HIV-1 budding occurs through glycolipid enriched microdomains termed lipid rafts leading to virion membranes enriched in cholesterol, sphingolipids, and raft associated proteins. The disruption of the lipid rafts significantly impairs HIV infectivity possibly at the fusion step. The accessory protein Nef had been proposed to increase viral budding through lipid rafts leading to the production of virions exhibiting increased infectivity. We previously showed that Nef enhances infectivity at an early step of HIV replication cycle leading to enhanced cytoplasmic delivery of viral particles to the cytoplasm. Using a recently developed HIV virion-based fusion assay, we tested the fusogenic properties of virions produced by cells with intact or disrupted lipid rafts and in presence or absence of Nef.

METHODS: We recently described a sensitive and specific HIV virion-based fusion assay involving the incorporation of β-lactamase-Vpr chimeras into virions and the detection of their fusion by β-lactamase cleavage of the fluorescent CCF2 dye loaded into the target cells. Using this assay, we compared the fusogenic properties of virions produced by cells where the lipid rafts were disrupted by incubation with hydroxypropyl-β-cyclodextrin (β-CD), or HIV virions produced from viral genomes containing or lacking the nef gene (HIV-wt or HIVΔNef).

RESULTS: We observed the following: 1) disruption of the lipid rafts in HIV producing cells by treatment with β-CD markedly diminished HIV fusion to target cells; 2) HIV-wt and HIVΔNef virions fused to target cells with comparable efficiencies; 3) the cholesterol content of the HIV-wt and HIVΔNef virions was indistinguishable; and 4) the decline in HIV virion infectivity observed following treatment of producer cells with β-CD was observed with both HIV-wt and HIVΔNef virions.

CONCLUSIONS: These findings demonstrate that fusion of HIV virions to target cells is markedly impaired when lipid rafts are disrupted in the producer cells, suggesting a possible facilitating role for cholesterol or other raft components in the fusion reaction. Further, the enhancement by Nef of viral infectivity cannot be explained by intrinsic differences in the fusogenic properties of HIV-wt and HIVΔNef virions. We are now exploring the possibility that Nef influences a subsequent step in the viral life cycle.

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