Involvement of Lipids in Different Steps of the Flavivirus Fusion Mechanism

Abstract

Flavivirus membrane fusion is triggered by acidic pH and mediated by the major envelope protein E. A structurally very similar fusion protein is found in alphaviruses, and these molecules are designated class II viral fusion proteins. In contrast to that of flaviviruses, however, alphavirus fusion has been shown to be absolutely dependent on the presence of cholesterol and sphingomyelin in the target membrane, suggesting significant differences in the fusion protein-membrane interactions that lead to fusion. With the flavivirus tick-borne encephalitis virus (TBEV), we have therefore conducted a study on the lipid requirements of viral fusion with liposomes and on the processes preceding fusion, specifically, the membrane-binding step and the fusion-associated oligomeric switch from E protein dimers to trimers. As with alphaviruses, cholesterol had a strong promoting effect on membrane binding and trimerization of the fusion protein, and-as shown by the use of cholesterol analogs-the underlying interactions involve the 3␤-hydroxyl group at C-3 in both viral systems. In contrast to alphaviruses, however, these effects are much less pronounced with respect to the overall fusion of TBEV and can only be demonstrated when fusion is slowed down by lowering the temperature. The data presented thus suggest the existence of structurally related interactions of the flavivirus and alphavirus fusion proteins with cholesterol in the molecular processes required for fusion but, at the same time, point to significant differences between the class II fusion machineries of these viruses.Entry of enveloped viruses into cells involves binding to specific receptors and fusion of the viral membrane with a cellular membrane. Both processes are controlled by viral surface glycoproteins. The proteins mediating fusion possess structural elements that interact with lipids in the target membrane, and in some cases, a specific lipid requirement has been demonstrated for these processes (reviewed in reference 16). The nature and specificity of these protein-lipid interactions, however, are still much less well understood than those of protein-mediated receptor binding.Most of the viral fusion proteins characterized so far can be divided into two classes (20). Class I comprises homotrimeric spike proteins that are synthesized as precursors and require proteolytic cleavage for their activation. They have aminoterminal or amino-proximal fusion peptides and form a coiledcoil postfusion structure. Such proteins are present in orthomyxoviruses, paramyxoviruses, retroviruses, and filoviruses (reviewed in references 34 and 43). In contrast, the class II viral fusion proteins of flaviviruses (E protein) and alphaviruses (E1 protein) are not spiky projections but are, instead, oriented parallel to the viral membrane and form an icosahedral network in the viral envelope. They do not appear to have the propensity to form alpha-helical coiled coils, they are not proteolytically cleaved but require the cleavage of a second, accessory protein for activatio...

Hepatitis C Virus Glycoproteins Mediate Low pH-dependent Membrane Fusion with Liposomes

“…SFV and Sindbis virus fusion depends strictly on the presence of cholesterol and sphingolipids in the target membrane (55,61). By contrast the overall fusion of TBEV with liposomes is only facilitated by the presence of cholesterol in target membranes (29), is minimally affected by cholesterol depletion from cell membranes, and sphingolipids have only a weak fusion-facilitating effect (33). The flexibility of our liposome/HCVpp system allows the precise evaluation of the influence of each lipid in the fusion reaction.…”

“…In the Flaviviridae family, it is known that low pH triggers glycoprotein conformational changes, leading to the formation of a fusion-competent trimer, in which the fusion peptide becomes exposed and inserts into the target (endosomal) membrane to initiate fusion. This process has been described for Flaviviruses such as TBEV (33) to be enhanced by the presence of cholesterol in the target membrane. Cholesterol has also been reported to enhance entry, but not the fusion process itself, of many other pH-dependent viruses (29).…”

Characterization of a Membrane-Associated Trimeric Low-pH-Induced Form of the Class II Viral Fusion Protein E from Tick-Borne Encephalitis Virus and Its Crystallization

“…As revealed by rate-zonal centrifugation in sucrose density gradients containing 0.8% n-OG (22), more than 90% of the material formed a defined peak at the position corresponding to an E trimer, and only a small amount (5 to 10%) sedimented as a dimer (Fig. 2A).…”

“…It can therefore be proposed that the strong protein interactions leading to these arrays are an important parameter for the membrane fusion event in both virus systems. Differences between flavi-and alphaviruses, however, exist in terms of the lipid dependence of membrane insertion, trimerization, and fusion (6,14,22,24). In contrast to alphaviruses, these processes in flaviviruses are not absolutely dependent on cholesterol and do not require sphingomyelin (6,22).…”

“…To convert the dimers to trimers, we then exposed them to acidic pH in the presence of large unilamellar liposomes, as described previously (21). Briefly, sE dimers were mixed with liposomes consisting of 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (PC), 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (PE), and 1-cholesterol (CH) (molar ratio, 1:1:2) at a ratio of 1 g of sE to 15 nmol of lipid (22). This mixture was acidified with 300 mM morpholineethanesulfonic acid (MES), incubated for 30 min at 37°C at pH 5.4, back neutralized, adjusted to 20% (wt/wt) sucrose in 20 mM triethanolamine (TEA) and 130 mM NaCl (pH 8.0) (TAN buffer), and was then applied to a 50% cushion, overlaid with 15% (wt/wt) sucrose and 5% (wt/wt) sucrose.…”