Session 36Poster Presentations Accessory Genes Session Day and Time: Tuesday 1:30 - 3:30 pm Room: Hall D
206 The Association of HIV-1 Nef with Lipid Rafts is Functionally Important for CD4 and MHC Class I Down-modulation Melissa Alexander Heaton*, Kodi S Ravichandran, Marie-Louise Hammarskjold, David Rekosh Univ of Virginia, Charlottesville
Background: Recently described lipid rafts are enriched in signaling proteins, which target there via saturated fatty acid additions, such as myristoylation and palmitoylation. HIV-1 Nef is myristoylated and reports have suggested that a fraction of Nef localizes to rafts.
Methods: We have addressed whether Nef targeting to rafts has functional significance by creating a Nef fusion protein (LAT-Nef) containing the N-terminal 35aa from the linker for activation of T-lymphocytes (LAT). Within this LAT sequence is a transmembrane domain followed by 2 cysteines at residues 26 and 29. LAT is palmitoylated at these cysteines, and this is critical for targeting the protein to rafts.
Results: When a plasmid expressing LAT-Nef was transfected into SupT1 cells, about 90% of the protein was found in the raft fraction. We also constructed a version of the LAT-Nef fusion protein that had the 2 cysteine residues mutated to alanine (LATC26,29A-Nef). About 5% of the mutant protein targeted to rafts. Both proteins were expressed at equal levels and targeted to the membrane fraction equivalently. The 2 fusion proteins were compared to each other, to wild-type Nef, and to non-myristoylated Nef for their ability to down-modulate cell surface MHC-I and CD4. LAT-Nef down-modulated MHC-I nearly as well as wt Nef. The mutant LATC26,29A-Nef was not as effective. The fluorescence intensity of surface MHC-I was reduced by LAT-Nef to 95% of the level of achieved by wt Nef, while LATC26,29A-Nef down-modulated MHC-I to less than 40% of that level. The results with CD4 were similar in that LAT-Nef down-modulated CD4 to a much greater extent than the mutant LATC26,29A-Nef. LAT-Nef was about 60% as efficient as wt Nef in CD4 down-modulation, while mutant LATC26,29A-Nef was 15% as efficient as wt Nef. Surprisingly, the non-myristoylated Nef showed a modest but dose-dependent CD4 down-modulation, although negligible activity was observed for MHC-I down-modulation.
Conclusions: Our results suggest that Nef targeting to lipid rafts is functionally significant for MHC-I and CD4 down-modulation. Since LAT-Nef works almost as well as wild-type Nef for MHC-I down-modulation, but less well than wild-type for CD4 down-modulation, raft involvement may be different in each of these processes.
