Studies of membrane fusion II. fusion of human erythrocytes by sendai virus

Abstract

ABSTRACT Thin section, negative stain and freeze-fracture electron microscopy have been used to study the fusion of human erythrocytes by Sendai virus. Sendai virus particles are shown to consist of an envelope covered with ∼ 12 nm-long projections or spikes which encloses a helical nucleocapsid. The spike complexes are dumbell-shaped and consist of 2 knobs, one ∼ 8 nm, the other ∼14 nm, in diameter connected by a narrow ∼ 3 nm-thick stalk, with the larger knob embedded in the lipid bilayer of the viral envelope. Virus particles bind to and agglutinate cells at 4 °C; fusion of viral envelopes with the erythrocyte membrane and extensive cell-cell fusion takes place following a brief incubation at 37 °C. At 4 °C most virus particles are roughly spherical and fractures through the viral envelope reveal concave E fracture faces with ∼ 14-nm-diameter intramembrane particles and convex P faces with a complementary arrangement of pits. Incubation at 37 °C results in a dramatic change in the structure of the viral envelope of many virus particles. Invaginations of the viral envelope give the virus a convoluted profile and such virus particles are characterized in freeze-fracture replicas by the presence of smooth linear ridges 30 nm wide and up to 0. ·5 μm long on E faces and by a complementary arrangement of linear grooves on P faces. Instead of ∼ 14 nm-diameter intramembrane particles on E faces, ∼ 8 nm particles are present on both P and E faces. These changes, which only take place when virus particles are bound to erythrocytes, are also accompanied by the disappearance of clearly defined surface spikes. Only virus particles having this altered morphology actually fuse with the erythrocyte membrane. The characteristic ‘ridged’ morphology of the viral envelope allows specific sites of viral envelope-cell fusion to be identified in freezefracture replicas and successive stages during the incorporation of the viral envelope to be reconstructed. These observations show that viral envelope-cell fusion is initiated by a cell- mediated temperature-sensitive change in the molecular organization of the viral envelope which allows the particle—denuded linear invaginations, possibly lipid bilayer, to interact and fuse with the erythrocyte membrane. Further observations suggest that cell-cell fusion does not occur directly between cells but is achieved by the simultaneous fusion of a virus particle with 2 adjacent erythrocytes.