Specific Roles for Lipids in Virus Fusion and Exit Examples from the Alphaviruses - Fusion of Biological Membranes and Related Problems - page 424

possibly lipids, may act to modulate the cholesterol dependence of SFV
fusion and exit.

4. THE ROLE OF SPHINGOLIPID IN ALPHAVIRUS FUSION

4.1. In Vitro Requirement for Sphingolipid in Virus-Membrane Fusion

Studies from the laboratory of Jan Wilschut have demonstrated that
the fusion of SFV with target liposomes is strikingly dependent on the pres-
ence of sphingolipid in the target membrane (Nieva et al., 1994; Wilschut et
al., 1995; Corver et al., 1995; Moesby et al., 1995; He et al., 1999). Both lipid
and content mixing assays demonstrated strong sphingolipid dependence,
indicating that sphingolipid is required for both hemifusion and com-
plete fusion. About 2 mole percent sphingomyelin is sufficient to support
maximal SFV liposome fusion (Nieva et al., 1994), considerably lower than
the ~30 mole percent cholesterol required for maximal SFV liposome
fusion (White and Helenius, 1980). Sphingolipid does not seem to be
required for the initial hydrophobic interaction of the virus with the target
membrane (Nieva et al., 1994; Moesby et al., 1995), while efficient virus-
liposome interaction does require cholesterol (Kielian and Helenius, 1984;
Wahlberg et al., 1992) (Table I). Recent experiments on SIN fusion with
liposomes indicate that this virus is also strongly sphingolipid dependent
(personal communication from J. Smit and J. Wilschut). Fusion studies with
a wide variety of sphingolipids have demonstrated an interesting require-
ment for distinct structural features of the sphingolipid molecule, as
detailed below. Thus both the cholesterol and sphingolipid requirements are
suggestive of specific lipid-protein interactions during fusion.

4.2. Structural Features of Fusion-Permissive Sphingolipids

Sphingomyelin, ceramide, and galactosyl ceramide are all active in SFV
fusion, indicating that the headgroup of the sphingolipid is not critical for
fusion and can be either phosphocholine, a hydroxyl group, or a carbohy-
drate moiety (Nieva et al., 1994). Ceramide is the minimal sphingolipid
that can support fusion, and the sphingosine base is inactive (Nieva et al.,
1994). The reaction is stereospecific for the naturally occurring D-erythro
stereoisomer while the three unnatural stereoisomers of ceramide are inac-
tive (Moesby et al., 1995). Acyl chain length does not appear critical since
both C18-ceramide and C8-ceramide are active (Corver et al., 1995; Moesby
et al., 1995). 3-deoxyceramide or 3-methoxyceramide do not support fusion,
demonstrating the importance of the 3-hydroxyl group on the sphingosine