Adaptation of Alphaviruses to Heparan Sulfate: Interaction of Sindbis and Semliki Forest Virus with Liposomes Containing Lipid-Conjugated Heparin

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycoaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 have been identified (E2:1; E2:70; E2:114), which mutate during adaptation and independently confer the ability to the virus to bind to cell-surface HS (W.B. Klimstra et al., J. Virol. 72:7357-7366, 1998). In this study, we used HS-adapted SIN mutants to evaluate a new model system, involving target liposomes containing heparin-conjugated phosphatidylethanolamine (HepPE) as an HS receptor analog for the virus. HSadapted SIN viruses, but not non-adapted wild-type SIN TR339, interacted efficiently with HepPE-containing liposomes at neutral pH. Binding was competitively inhibited by soluble heparin. Despite the efficient binding of HSadapted SIN viruses to HepPE-containing liposomes at neutral pH, there was no fusion under these conditions. Fusion did occur, however, at low pH, consistent with cellular entry of the virus via acidic endosomes. At low pH, wild-type or HSadapted SIN viruses underwent fusion with liposomes with or without HepPE with similar kinetics, suggesting that interaction with the HS receptor analog at neutral pH has little influence on subsequent fusion of SIN at acidic pH. Finally, Semliki Forest virus (SFV), passaged frequently on BHK-21 cells, also interacted efficiently with HepPE-containing liposomes, indicating that SFV, like other alphaviruses, readily adapts to cell-surface HS. In conclusion, the liposomal model system presented in this paper may serve as a novel tool to study receptor interactions and membrane fusion properties of HS-interacting enveloped viruses. Heparan sulfate interaction and membrane fusion of alphaviruses 55