257 Heavy Rafts: A New Class of Rafts and Its Possible Role in HIV Assembly D. Hammache *1, S. Alais 1, M. Louwagie 2, J.Garin2, D.Gerlier1 1Ctr Natl de la Res Sci UMR 5537, Nice, France and 2INSERM-CEA
Background: Assembly of several enveloped viruses, including HIV, occurs within membrane regions rich in glycosphingolipids and cholesterol called membrane rafts. Rafts can be isolated by flotation on a sucrose gradient, as low-density, Triton X-100 insoluble membrane. We recently observed that some membrane-like materials can be also recovered from the high density sucrose fractions. We have undergone the characterization of this material and of its contents in HIV-1 proteins.
Methods: Low density and "heavy rafts" microdomains were isolated from cell lines expressing HIV Pr55gag lysed in cold 1% Triton X-100. Lipids were analyzed by HPTLC. Proteins were analyzed by Western blot and/or proteomic analysis by mass spectrometry.
Results: The existence of new microdomains of plasma membrane, called "heavy rafts," is proposed on the following criteria: 1) insolubility in 1% Triton X-100 at 4°C; 2) solubility after cholesterol depletion; 3) solubility in Octyl-D glucoside; 4) glycosphingolipids + cholesterol contents; 5) calveolin contents; and 6) cell surface location for at least in part. A larger amount of HIV-1 Gag is associated with "heavy rafts" than the classical low density rafts. The proteomic analysis of "heavy rafts" reveals the presence of several cellular proteins previously reported 1) to be localized inside the virus (actin, Glyceraldehyde 3-PhosphoDesydrogenase); 2) to interact with virus p24CA and p17MA protein (translation factor elongation 1a); 3) to assist Env protein folding (BiP, calnexin); 4) to be involved in HIV induced syncytia formation (CD98); or 5) to interact with HIV-1 Tat and Rev protein (nucleophosmin).
Conclusions: We propose that the "heavy rafts" are a potential location for the assembly of HIV-1. Further study of the interaction between cellular and HIV-1 proteins within the "heavy rafts" may provide a more complete understanding of mechanism of assembly and budding of HIV-1.
