265 HIV-1 Gag Alters Actin Polymerization and Increases T-cell Chemotaxis FJ Ibarrondo*, P Krogstad, YZ Geng, G Baldwin David Geffen Sch of Med at UCLA, Los Angeles, CA
Background: The HIV-1 Gag protein interacts with filamentous actin in vitro and in vivo, but the significance has remained elusive. Based on prior studies involving human and nonhuman cell types, we hypothesized that these interactions might alter the turnover of the actin cytoskeleton of lymphocytes, leading to enhanced motility and dissemination of infected cells.
Methods: Gag effects on motility were examined by transfecting Jurkat T-cells with a vector that allows Rev-independent expression of HIV-1 Gag. Transmembrane motility assays were performed, using SDF 1Alpha as a chemo attractant, and flow cytometry was used to examine CXCR4 expression and the motility of Gag-positive cells. Gag effects on microfilament turnover were examined by treating cells with latrunculin, which promotes actin filament disassembly, and monitoring filament recovery by flow cytometry after the drug was washed out. Recombinant Gag was prepared in E. coli as a fusion protein with a S. cereviseae intein and purified by affinity chromatography with DTT mediated cleavage of the fusion tag. Activation of ERK and Rho, central signaling factors in cytoskeleton remodeling, was examined by immunoprecipitation. The direct effects of Gag on actin filament assembly were examined in vitro polymerization assays using highly purified rabbit actin.
Results: HIV-1 Gag distributed to cytoskeletal fractions containing long actin filaments and co-localized with the pool of short actin filaments that are mobilized in actin-based motility. HIV-1 Gag expression did not alter the cell surface expression of CXCR4, but significantly increased the transmembrane chemotactic motility of Jurkat T-cells in response to its ligand, SDF 1Alpha. Gag did not appear to alter signal transduction in the Jurkat T-cells (no change in amount of activated Rho or ERK). Gag enhanced the turnover of actin filaments in permeabilized T-cells, but not did not alter in vitro actin polymerization kinetics.
Conclusions: Gag expression modifies the polymerization-depolymerization equilibrium of microfilaments, and is associated with augmented chemotactic motility. This may be mediated by other actin-binding proteins, since Gag did not alter actin assembly in in vitro assays. Gag enhancement of cell motility could contribute to the dissemination of HIV-infected cells.
