Recent studies have suggested the plasma membrane contains cholesterol-enriched microdomains known as lipid rafts. Gag multimerization presumably as a component of liquid-ordered membrane microdomains. To our knowledge, this is the 1st report showing that membrane cholesterol facilitates the connection of a myristylated protein with the cytoplasmic leaflet of the plasma membrane. An increasing number of enveloped and non-enveloped viruses are proposed to associate with lipid rafts during assembly (Briggs et al., 2003; Ono and Freed, 2005; Suomalainen, 2002). Analysis of the relationships between cholesterol-enriched lipid rafts and particle production mediated by these viruses will likely elucidate common aspects of membrane microdomains that are exploited by diverse viruses. Materials and Methods Plasmids Molecular clones encoding Gag derivatives, pNL4-3/Fyn(10)fullMA and pNL4-3/Fyn(10)MA/delNC (Ono et al., 2004), or an inactive PR [pNL4-3/PR?, (Huang et al., 1995)] were described previously. A PR? version of pNL4-3/Fyn(10)fullMA [pNL4-3/Fyn(10)fullMA /PR?] was LY317615 distributor constructed by introducing the SphI-to-EcoRI fragment (pNL4-3 nt 1443-5743) from pNL4-3/PR? into pNL4-3/Fyn(10)fullMA. Construction of pCMVNLGagPolRRE using pCMVGagPolRRE [a kind gift from D. Rekosh, University of Virginia (Srinivasakumar et al., 1997)] was described previously (Ono and Freed, 2001). The VSV-G expression vector pHCMV-G (Yee et al., 1994) was generously provided by J. Burns (University of California, San Diego). An expression plasmid encoding FynGFP (van’t Hof and Resh, 1997) was kindly provided by M. LY317615 distributor Resh (Memorial Sloan-Kettering Cancer Center). Cells, transfections, and infections HeLa cells were cultured as previously described (Freed and Martin, 1994). Gag was expressed either by transfecting cells with molecular clones or by infecting with high-titer vector virus stocks. Transfection of LY317615 distributor HeLa cells was performed by the calcium phosphate method as previously described (Freed and Martin, 1994) or by using Lipofectamine 2000 (Invitrogen) according to LY317615 distributor the manufacturer’s instructions. Infection of HeLa cells with virus stocks pseudotyped with VSV-G was performed as described previously (Ono et al., 2005). VSV-G-pseudotyped virus stocks were prepared by transfecting HeLa cells with pCMVNLGagPolRRE, pHCMV-G, and the molecular clones pNL4-3/PR?. Cholesterol depletion, metabolic labeling, and immunoprecipitation Depletion of cholesterol and metabolic labeling were performed as described previously (Ono and Freed, 2001). Briefly, HeLa cells were cultured in Met?/Cys? RPMI-1640 supplemented with 2% cholesterol-depleted serum LY317615 distributor (CDS) in the presence of 10 mM MCD for 30 min. Subsequently, cells were metabolically labeled with [35S]Met/Cys Rabbit Polyclonal to ADAM 17 (Cleaved-Arg215) in Met?/Cys? RPMI-1640 supplemented with 2% CDS for 2 h for the analyses of virus release efficiency. Preparation of cell lysates, pelleting of virions in the ultracentrifuge, and immunoprecipitation of cell- and virion-associated proteins with HIV-Ig (obtained from the NIH AIDS Research and Reference Reagent Program) was detailed previously (Freed and Martin, 1994). Quantification of immunoprecipitated proteins was performed by phosphorimager analysis. Virus release efficiency was calculated as the amount of virion-associated Gag as a fraction of total (cell plus virion) Gag synthesized during a 2-h labeling period. For membrane binding analyses, MCD-treated cells were pulse labeled with [35S]Met/Cys in Met?/Cys? RPMI-1640 supplemented with 2% CDS for 5 min and chased in DMEM containing 5% CDS for 15 min. Alternatively, cells were cultured for 2 days in DMEM containing 5% CDS in the presence of 2 M simvastatin and 500 M mevalonate. After starving in RPMI-Met?/Cys? medium containing 2 M simvastatin and 500 M mevalonate for 30 min, cells were labeled in the same medium with [35S]Met/Cys for 5 min followed by a 15-min chase period. Membrane binding analyses and denaturation of Gag Analyses of Gag-membrane binding were performed as previously described (Ono et al., 2000a; Ono and Freed, 1999; Ono and Freed, 2001; Ono et al., 2005). Denaturation of Gag was performed as described previously (Ono et al., 2005). Immunofluorescence microscopy Fixation, permeabilization, and immunostaining of transfected HeLa cells were performed as previously described (Ono et al., 2004;.