Session 4Oral Abstract Presentations HIV Replication: Entry and Assembly Session Day and Time: Tuesday 10 am - 12:30 pm Presentation Time: 10:00 Room: Ballroom A
17 Inside-out Regulation of HIV-1 Particle Fusion Christopher Aiken*1, Donald J. Wyma1, Janet E. Lineberger2, Michael D. Miller2 1Vanderbilt Univ Sch of Med, Nashville, TN and 2Merck Res Labs, West Point, PA
Background: Cell entry of human immunodeficiency virus type 1 (HIV-1) particles requires a functional glycoprotein complex composed of the attachment protein gp120 and the fusion protein gp41. Proteolytic maturation of the viral core is necessary for completion of early post-entry steps in infection. Because entry of immature virions would be predicted to result in nonproductive infection, we hypothesized that the fusion activity of the HIV-1 envelope glycoprotein complex is functionally linked to HIV-1 core maturation.
Methods: We used a novel fluorescence-based virus-cell reporter assay to quantify the fusion of HIV-1 particles arrested at various stages of core maturation. The assay employs HIV-1 particles that have incorporated beta-lactamase via fusion to Vpr. Fusion of the particles with target cells results in delivery of active beta-lactamase into target cells, resulting in cleavage of the fluorescent substrate CCF2/AM and a resulting increase in blue fluorescence.
Results: Immature HIV-1 particles were markedly impaired for fusion with T-cells. Truncation of the gp41 cytoplasmic tail, pseudo-typing by VSV-G, or cleavage of the viral core precursor protein Pr55Gag between the MA and NC domains, rescued the fusion defect.
Conclusions: We have identified a novel mechanism for regulating HIV-1 entry through coupling membrane fusion activity to core maturation via binding of the gp41 cytoplasmic domain to Pr55Gag. These findings have implications for therapeutic strategies targeting HIV-1 entry and for HIV-1 vaccine development.
