Spin-label study on fusion of red blood cells induced by hemagglutinating virus of Japan

Abstract

Fusion of red blood cells (RBC) induced by hemagglutinating virus of Japan (HVJ) has been studied using a phosphatidylcholine spin label. The spin label was readily incorporated and diffused into the lipid bilayer portion of the viral envelope. The exchange broadening in the electron spin resonance (ESR) spectrum of densely labeled virus disappeared rapidly when the virus was mixed with RBC at 37 degrees. The spectrum gradually approached that of the host cell spin labeled with the phosphatidylcholine label. The results directly indicate transfer and intermixing of phospholipid molecules between the viral envelope and RBC membrane. The transfer reaction was strongly dependent on temperature. No transfer was observed at lower temperatures where the virus adsorbed to the cell and caused aggregation but no hemolysis and fusion. The transfer rate remained negligibly small until 19 degrees and increased rapidly between 25 and 30 degrees. The virus-induced hemolysis showed similar temperature dependence. The transfer rate was greatly reduced under inhibitory conditions of fusion: glutaraldehyde treatment of RBC, trypsin treatment of HVJ, or the presence of concanavalin A. Only slight transfer was observed from fusion-inactive influenza virus to RBC. The transfer was greatly enhanced by the help of HVJ. The close parallelism suggests that the transfer and intermixing are necessary steps to the cell fusion. The transfer rate was dependent on fluidity of the host cell membrane and independent of the viral dose. The virus-induced transfer of phospholipid molecules between RBC's was also detected by the spin label. Its temperature dependence was quite similar to that for the virus-to-cell transfer. The intercellular transfer was nearly proportional to the viral dose.

Changes in cell membrane microviscosity associated with adsorption of viruses Differences between fusing and non‐fusing viruses

“…It is probable that rearrangement of the virus and cell proteins in the cell membrane might be held responsible for the observed changes described here, in our previous studies [l] , as well as in other investigations [6,7,18] .…”

“…Such a clustering would obviously change the existing protein lipid interaction in the plane of the membrane. Additional evidence supporting the hypothesis that viruses induce movement of integral proteins in the membrane is the observation [l] as well as that of [7] that at temperatures below 10°C the viruses, though adsorbing normally, do not affect the fluidity of the phospholipids in the cell membrane.…”

“…1,2, [6] ). This effect may be explained by an assumption that during the process of fusion of the virus envelope with the cell membrane their respective lipids intermix [7,8]. Paramyxoviruses penetrate their host cells by a process in which the first step, following adsorption, is the fusion of the viral envelope with the cell membrane [9].…”

“…The paradoxical finding that the rapid movement and clustering of intramembranal particles induced by paramyxoviruses [7,17] occurs at the same time as the increase in the microviscosity of the host cell phospholipids can be interpreted by assuming that these two chains of events are not causally linked, but produced by two independent mechanisms.…”

Gene transfection by quantitatively reconstituted Sendai envelope proteins into liposomes

“…It has been well documented that the F glycoprotein of Sendai virus promotes the virus -mediated membrane fusion even though the precise mechanism for the process has not been clarified yet. 21,22 Also, it has been considered that the Sendai HN glycoprotein serves as an anchoring protein, specifically attaching to sialoglycoproteins and sialoglycolipids in cell membranes. In addition to the anchoring function, some scientists proposed that the HN glycoprotein also plays an active role in the fusion process.…”