Sphingolipid and Cholesterol Dependence of Alphavirus Membrane Fusion: LACK OF CORRELATION WITH LIPID RAFT FORMATION IN TARGET LIPOSOMES

Barry-Lee Waarts,Robert Bittman,Jan Wilschut

doi:10.1074/jbc.m206998200

Barry-Lee Waarts, Robert Bittman + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m206998200

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Abstract

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes. Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane. This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or "lipid rafts," in the membrane fusion and cell entry process of the virus. In this study, large unilamellar vesicles (LUVs) were prepared from synthetic sphingolipids and sterols that vary with respect to their capacity to promote microdomain formation, as assessed by gradient flotation analysis in the presence of Triton X-100. SFV and SIN fused with LUVs irrespective of the presence or absence of Triton X-100-insoluble microdomains. These results suggest that SFV and SIN do not require the presence of lipid rafts for fusion with target membranes. Furthermore, it is not necessary for sphingolipids to reside in a detergent-insoluble complex with cholesterol to promote SFV or SIN fusion.

Highlights

Semliki Forest virus (SFV) and Sindbis virus (SIN) are enveloped viruses that infect their host cells by receptor-mediated endocytosis and subsequent fusion from within acidic endosomes
Evaluation of the Presence of detergent-insoluble complexes (DICs) in large unilamellar vesicles (LUVs)—To evaluate the presence of DICs in liposomes, LUVs consisting of PC/PE/SPM/ Chol were treated with Triton X-100 at 4 °C and subjected to a density gradient flotation analysis, as described under “Experimental Procedures.”
About 90% of the phospholipid in the DIC-containing fractions isolated from PC/PE/SPM/Chol/LUVs consisted of SPM, as determined by phospholipid analysis of the lipid spots separated by thin-layer chromatography (TLC) (Fig. 2)

Summary

LACK OF CORRELATION WITH LIPID RAFT FORMATION IN TARGET LIPOSOMES*

Fusion of the viral envelope requires the presence of both cholesterol and sphingolipids in the target membrane This is suggestive of a possible involvement of sphingolipid-cholesterol microdomains, or “lipid rafts,” in the membrane fusion and cell entry process of the virus. The dependence of SFV and SIN fusion on the presence of both Chol and sphingolipids in the target membrane is suggestive of a possible involvement of sphingolipid-Chol microdomains or “lipid rafts,” which are present in mammalian cellular membranes as small entities (21, 22) These lipid rafts are thought to exist in bilayers in the liquid-ordered Lo phase as complexes consisting of Chol and (glyco)sphingolipids with predominantly saturated acyl chains (23). HIV-1 virions pass a barrier of mucosal epithelial cells by a process called transcytosis, which requires the presence of galactosylceramide in lipid rafts to mediate virion binding (34)

To address the question as to whether lipid rafts are involved

EXPERIMENTAL PROCEDURES

RESULTS

Phospholipid in DICs

DISCUSSION