[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: Inherent to their condition of obligate intracellular parasites, viruses rely on hijacking of cell host machinery for multiplication. This dependence on cellular factors also includes the use of cellular proteins and lipids for key steps of the viral life cycle, i.e. entry into the cell, genome replication, assembly, and release of progeny virus particles. Viruses can take advantage of cellular lipids as entry receptors, cofactors for membrane fusion, scaffolding molecules for replication complex assembly, and even as structural components of viral particles (especially in the case of enveloped viruses). Indeed, the relationship between viral infection and cellular lipids goes beyond a mere dependence on specific lipids for viral multiplication. Recent advances have uncovered a fine tuned intimate connection between lipid metabolism and viral infection. These studies have unveiled that viruses orchestrate profound alterations of cellular lipid metabolism that favour the accumulation of specific metabolites for their own purposes. For a wide variety of viruses this implies that infected cells undergo specific intracellular membrane rearrangements to build up virus-induced organelle-like structures that provide the adequate platforms for viral replication, which can also contribute to evasion of the innate immune response. Key roles of cellular lipids like cholesterol, fatty acids, or specific phospholipids have been related to these processes. To build up this specific microenvironment viruses co-opt cell host factors involved in membrane remodelling and lipid synthesis. The strong dependence of viral infection on specific lipids and cellular factors involved in their metabolism opens new lines of investigation for the search and development of alternative new antiviral strategies targeting lipid metabolism to interfere with virus multiplication.
[show abstract][hide abstract] ABSTRACT: Sec9p and Spo20p are two SNAP25 family SNARE proteins specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate intracellular trafficking between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires enzymatically active Spo14p, which is a phosphatidylcholine (PC)-specific phospholipase D that hydrolyzes PC to generate phosphatidic acid (PA). Phosphatidic acid is required to localize Spo20p properly during sporulation; however, it seems to have additional roles that are not fully understood. Here we compared the fusion mediated by all combinations of the Sec9p or Spo20p C-terminal domains with Sso1p/Sso2p and Snc1p/Snc2p. Our results show that Spo20p forms a less efficient SNARE complex than Sec9p. The combination of Sso2p/Spo20c is the least fusogenic t-SNARE complex. Incorporation of PA in the lipid bilayer stimulates SNARE-mediated membrane fusion by all t-SNARE complexes, likely by decreasing the energetic barrier during membrane merger. This effect may allow the weak SNARE complex containing Spo20p to function during sporulation. In addition, PA can directly interact with the juxtamembrane region of Sso1p, which contributes to the stimulatory effects of PA on membrane fusion. Our results suggest that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions may be coordinately regulated to control the rate and specificity of membrane fusion.
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