Lipids in viral fusion

Laboratory of Experimental and Computational Biology, NCI-FCRDC, Frederick, MD, USA.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2002; 199:61-81. DOI: 10.1385/1-59259-175-2:61
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Available from: Anu Puri, Jan 08, 2015
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    • "Cell-cell fusion between CD4 + targets and gp120-gp41-expressing HeLa cells was assayed using a fluorescent dye transfer method [28]. We used the fluorescent dyes CMTMR (green emission, ex/em 540/566 nm), CMFDA (blue emission, ex/em 492/516 nm) or Calcein (blue emission, ex/em 496/517 nm) to label the cells for fusion assay. "
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    ABSTRACT: We investigated the effect of receptor mobility on HIV-1 envelope glycoprotein (Env)-triggered fusion using B16 mouse melanoma cells that are engineered to express CD4 and CXCR4 or CCR5. These engineered cells are resistant to fusion mediated CD4-dependent HIV-1 envelope glycoprotein. Receptor mobility was measured by fluorescence recovery after photobleaching (FRAP) using either fluorescently-labeled antibodies or transient expression of GFP-tagged receptors in the cells. No significant differences between B16 and NIH3T3 (fusion-permissive) cells were seen in lateral mobility of CCR5 or lipid probes. By contrast CD4 mobility in B16 cells was about seven-fold reduced compared to its mobility in fusion-permissive NIH3T3 cells. However, a CD4 mutant (RA5) that localizes to non-raft membrane microdomains exhibited a three-fold increased mobility in B16 cells as compared with WT-CD4. Interestingly, the B16 cells expressing the RA5 mutant (but not the wild type CD4) and coreceptors supported HIV-1 Env-mediated fusion. Our data demonstrate that the lateral mobility of CD4 is an important determinant of HIV-1 fusion/entry.
    Full-text · Article · Feb 2008 · Molecular Membrane Biology
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    • "Cell – cell fusion between CD4 + targets and HIV-1 Envexpressing effector cells was assayed using the fluorescent dye-transfer method (Puri et al., 2002). We used the fluorescent dyes CMTMR (green emission, ex/em 540/566 nm), CMFDA (blue emission, ex/em 492/516 nm), or Calcein (blue emission, ex/em 496/517 nm) to label the cells for fusion assay. "
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    ABSTRACT: We had previously reported that glycosphingolipids (GSL) support human immunodeficiency virus type 1 (HIV-1) entry. In this study, we further examined this issue by expressing HIV-1 receptors in GSL-deficient GM95 cells. GM95 cells expressing low levels of CD4 and CXCR4 or CCR5 did not support HIV-1 Env-mediated fusion. However, higher expression of these receptors rendered GM95 cells highly susceptible to fusion with cells expressing appropriate HIV-1 envelope glycoproteins (HIV-1 Envs). The GM95 cells exhibited a different fusion phenotype when compared with GSL(+) NIH3T3 cells bearing similar receptor levels. Fusion of GM95 targets expressing higher levels of CD4 and coreceptors occurred at 25 degrees C and was sensitive to cholesterol depletion or disruption of the cytoskeleton. In contrast, the fusion threshold of NIH3T3CD4X4/R5 targets was at >/=28 degrees C as previously reported and was insensitive to cholesterol depletion or cytoskeletal network disruption. On the basis of these observations, we propose that target membrane GSLs support HIV-1 Env-mediated fusion at low density of receptors by stabilizing receptor pools in natural targets.
    Full-text · Article · Jan 2004 · Virology
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    ABSTRACT: HIV-1 uses CD4 and chemokine receptors to enter cells. However, other target membrane components may also be involved. This study examines the role of glycosphingolipids (GSL) in HIV-1 entry into primary lymphocytes and its modulation by an inhibitor of GSL biosynthesis. CD4 lymphocytes purified from normal or the p-group subtype individuals that were defective in Gb3 synthesis were treated with a GSL biosynthesis inhibitor, 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP). The PPMP-treated cells were tested for HIV-1 replication by measuring p24 antigen production for 7-14 days post-infection and for susceptibility to HIV-1 Env-mediated fusion monitored by a fluorescent dye transfer assay. The effects of PPMP treatment on HIV-1 binding to CD4 lymphocytes were also examined by measuring HIV-1 p24. CD4 lymphocytes from p donors that are devoid of Gb3, but have elevated levels of GM3 were highly susceptible to HIV-1 fusion/entry. Pre-treatment of primary human CD4 lymphocytes from normal or p-sub-group type with PPMP, significantly reduced HIV-1 replication with no change in CD4 and CXCR4 levels. Inhibition of HIV-1 infection was due to the block in HIV-1 Env-mediated plasma membrane fusion. Binding of HIV-1 to CD4 lymphocytes was not affected by PPMP treatment. Manipulation of glycosphingolipid metabolic pathways may alter susceptibility of CD4 lymphocytes to HIV-1 entry.
    Full-text · Article · May 2004 · AIDS
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