Adaptation of alphaviruses to heparan sulfate: interaction of Sindbis and Semliki forest viruses with liposomes containing lipid-conjugated heparin

Abstract

Passage of Sindbis virus (SIN) in BHK-21 cells has been shown to select for virus mutants with high affinity for the glycosaminoglycan heparan sulfate (HS). Three loci in the viral spike protein E2 (E2:1, E2:70, and E2:114) have been identified that mutate during adaptation and independently confer on the virus the ability to bind to cell surface HS (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, 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 lipid-conjugated heparin (HepPE) as an HS receptor analog for the virus. HS-adapted SIN, but not nonadapted 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 HS-adapted SIN 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 HS-adapted SIN 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 low 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 for the study of receptor interactions and membrane fusion properties of HS-interacting enveloped viruses.

FIG. 1.

Binding of HS-adapted SIN 3970 to HepPE-containing liposomes. [³⁵S]methionine-labeled SIN (approximately 10⁸ to 10⁹ virus particles) was incubated with HepPE-supplemented PC-PE-SPM-Chol liposomes (100 μM liposomal phospholipid) at pH 7.4 for 1 h at4°C. Binding was determined by flotation analysis on sucrose density gradients as described in Materials and Methods. (A) Gradient profiles obtained after incubation of virus either with control liposomes lacking HepPE (squares) or with liposomes supplemented with 0.01 mol% HepPE (diamonds) or 0.02 mol% HepPE (circles). (B) Extents of binding of SIN 3970 to HepPE-containing liposomes as a function of the molar ratio of HepPE to total phospholipid in the liposomes. Results are averages of triplicate binding measurements.

FIG. 2.

Binding of HS-adapted SIN 3970 or TRSB and nonadapted SIN TR339 to liposomes supplemented with 0.01 mol% HepPE. The extent of binding was determined as described in the legend to Fig. 1, unless indicated otherwise. (A) Binding for 1 h at 4°C. Solid bars, PC-PE-SPM-Chol liposomes without HepPE; shaded bars, PC-PE-SPM-Chol liposomes supplemented with 0.01 mol% HepPE. (B) Binding under the conditions indicated. Open bars, TR339; hatched bars, 3970; crosshatched bars, TRSB. Bars represent averages of triplicate binding measurements.

FIG. 3.

Effect of soluble heparin on binding of HS-adapted SIN 3970 to PC-PE-SPM-Chol liposomes supplemented with 0.01 mol% HepPE; incubation took place at pH 7.4 for 1 h at 4°C. Binding was determined as described in the legend to Fig. 1. Squares, 5 mg of soluble heparin/ml; circles, 1 mg of soluble heparin/ml; diamonds, control without soluble heparin.

FIG. 4.

Low-pH-dependent fusion of HS-adapted SIN mutants with HepPE-containing liposomes. Online fusion experiments were performed at 37°C, as described in Materials and Methods. The final virus and liposome concentrations were 1.0 and 100 μM (membrane phospholipid), respectively, unless indicated otherwise. (A) Fusion curves of SIN with PC-PE-SPM-Chol liposomes, supplemented with 0.01 mol% HepPE, at pH 5.0 or pH 7.4. Curves a, TR339; curves b, TRSB; curves c, 3970. (B) Fusion at pH 5.0 of SIN 3970 with liposomes containing 0.01 mol% HepPE after isolation of virus-liposome complexes by flotation on a sucrose density gradient, essentially as in Fig. 1, except that pyrene-labeled virus was used. Peak fractions were collected from the top of the gradient and, after appropriate dilution in HNE, acidified to pH 5.0 at 37°C. Curve a, fusion of isolated virus-liposomes complexes; curve b, fusion of the initial mixture of virus and HepPE-containing liposomes at 1.0 and 100 μM (membrane phospholipid), respectively, before flotation on the sucrose density gradient.

FIG. 5.

Kinetics of low-pH-dependent fusion of HS-adapted SIN with HepPE-containing or control liposomes. Online fusion experiments were performed at 37°C, as described in the legend to Fig. 4, at final virus and liposome concentrations of 1.0 and 100 μM (membrane phospholipid), respectively. The initial rate of fusion as a function of pH was determined from the tangents to the first parts of the fusion curves. Squares, 3970; circles, TRSB; diamonds, TR339. (A) Liposomes supplemented with 0.01 mol% HepPE; (B) control PC-PE-SPM-Chol liposomes without HepPE. All fusion measurements were repeated at least three times.

FIG. 6.

Fusion of pyrene-labeled SIN 3970 with liposomes containing photoChol at pH 5.0. Fusion was measured online at 37°C as described in the legend to Fig. 4. Curve a, PC-PE-SPM-photoChol liposomes supplemented with 0.01 mol% HepPE; curve b, PC-PE-SPM-photoChol liposomes without HepPE; curve c, PC-PE-SPM-Chol liposomes supplemented with 0.01 mol% HepPE; curve d, PC-PE-SPM-Chol liposomes without HepPE. All fusion measurements were repeated at least three times.

FIG. 7.

Fusion of SIN with liposomes, assayed as the degradation of viral capsid protein by liposome-encapsulated trypsin. [³⁵S]methionine-labeled SIN (approximately 10⁸ to 10⁹ virus particles) was incubated with trypsin-containing PC-PE-SPM-Chol liposomes supple-mented with 0.01 mol% HepPE (100 μM liposomal phospholipid) at 37°C, and viral capsid protein degradation was determined as described in Materials and Methods. (A and B) Results for HS-adapted SIN 3970 (A) and nonadapted SIN TR339 (B) with either trypsin-containing liposomes (lanes a and d), empty liposomes (lanes b and e), or trypsin-containing liposomes in the presence of Triton X-100 and absence of a trypsin inhibitor in the medium (lanes c and f) at either pH 7.4 (lanes a to c) or pH 5.0 (lanes d to f). (C and D) Quantification of the extent of capsid protein degradation with liposomes supplemented with 0.01 mol% HepPE (C) or with control liposomes without HepPE (D). Open bars, TR339; hatched bars, 3970; crosshatched bars, TRSB. All capsid degradation experiments were repeated at least twice.

FIG. 8.

Interaction of SFV with BHK-21 cells, heparin-agarose beads, and HepPE-containing liposomes. (A) [³⁵S]methionine-labeled SFV particles (approximately 10⁸ to 10⁹ virus particles) were added to BHK-21 cell monolayers or heparin- or albumin-agarose beads, and binding was measured after incubation for 1 h at 4°C, as described in Materials and Methods. Bar a, binding to BHK-21 cells; bar b, binding to heparin-agarose beads; bar c, binding to albumin-agarose beads. (B) Binding of [³⁵S]methionine-labeled SFV (approximately 10⁸ to 10⁹ virus particles) to PC-PE-SPM-Chol liposomes supplemented with various concentrations of HepPE (100 μM liposomal phospholipid) during incubation at pH 7.4 for 1 h at 4°C. Binding of SFV to liposomes was assessed as described in the legend to Fig. 1. Each bar represents the average of triplicate binding measurements.

FIG. 9.

Low-pH-dependent fusion of pyrene-labeled SFV with liposomes. Fusion of pyrene-labeled SFV with PC-PE-SPM-Chol liposomes with or without 0.01 mol% HepPE (100 μM liposomal phospholipid) at 37°C was determined at pH 5.5 or pH 7.4, essentially as described in the legend to Fig. 4. Curves a and c, PC-PE-SPM-Chol liposomes supplemented with 0.01 mol% HepPE; curves b and d, PC-PE-SPM-Chol liposomes without HepPE. All fusion measurements were repeated at least three times.