Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.

Lateral motion of the viral envelope proteins in the target cell membrane was shown recently to be essential for cell fusion by Sendai virus (Henis, Y. I., Herman-Barhom, Y., Aroeti, B., and Gutman, O. (1989) J. Biol. Chem. 264, 17119-17125). To explore the mechanism that gives rise to this requirement, we have now investigated the distribution of Sendai virus envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) on human erythrocytes in the course of fusion, using fluorescence microscopy and image analysis. In these studies, both the F and the HN proteins were found to accumulate in cell-cell contact regions, on the time scale of the fusion process. We propose that migration of the viral glycoproteins to cell contact regions and accumulation at the contact sites are essential parts of the fusion mechanism and form the basis to the requirement for their lateral motion in the fusion event.

The rotational mobility of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase) was determined by using erythrosin (ER)-labeled monovalent Fab' antibody fragments directed specifically against either F or HN. By use of time-resolved phosphorescence anisotropy, the rotational mobility of Er-Fab'-viral glycoprotein complexes was studied both in the envelopes of unfused virions bound to erythrocyte ghosts and in the target cell membrane after fusion had occurred. The rotational correlation times (phi) of Er-Fab'-labeled F and HN were rather similar in the envelopes of bound unfused virions, but highly different in membranes of fused cells. The different phi values indicate that F and HN diffuse separately in the target cell membrane and for the major part are not complexed together. The temperature dependence of the phi values of the Er-Fab'-viral glycoprotein complexes revealed a breakpoint at 22 degrees C for the F protein both in bound virions and in the membranes of fused cells, and for the HN proteins in the envelopes of bound virions. In all these cases, the phi values increased between 4 and 22 degrees C, demonstrating a reduction in the rate of rotational diffusion. Further elevation of the temperature reversed the direction of the change in phi. This phenomenon may reflect a temperature-dependent microaggregation of F and HN saturating at ca. 22 degrees C and presumably related to the fusion mechanism since the breakpoint temperature correlates closely with the threshold temperature for virus-cell and cell-cell fusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Fusion of human erythrocytes by Sendai virions is accompanied by lateral mobilization of the viral envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) in the target cell membrane; the dynamic parameters characterizing the lateral diffusion of F and HN in the fused cell membrane are identical [Henis, Y. I., & Gutman, O. (1987) Biochemistry 26, 812-819; Aroeti, B., & Henis, Y. I. (1988) Biochemistry 27, 5654-5661]. This identity raised the possibility that F and HN diffuse together in the cell membrane in mutual heterocomplexes. In order to investigate the possible formation of F-HN complexes in the target cell membrane, which could be important for the fusion process mediated by the viral envelope proteins, we combined fluorescence photobleaching recovery (FPR) measurements of the lateral mobility of the viral glycoproteins with antibody-mediated cross-linking of F or HN. After fusion, one viral glycoprotein type was immobilized by cross-linking with highly specific bivalent polyclonal IgG. The other glycoprotein type was labeled with fluorescence monovalent Fab' fragments that do not induce cross-linking, and its mobility was measured by FPR. Neither the mobile fraction nor the lateral diffusion coefficient of the Fab'-labeled viral glycoproteins was affected by immobilization of the second viral envelope protein, demonstrating that F and HN diffuse independently in the target cell membrane and are not associated in mutual complexes.

Fluorescence photobleaching recovery was employed to study the effects of specific immobilization of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin-neuraminidase) on the virally mediated fusion of human erythrocytes. Lateral immobilization of varying fractions of F and/or HN (after virus adsorption and hemagglutination, but before fusion) was achieved by cross-linking them with succinyl concanavalin A (inhibiting both F and HN) or with specific rabbit IgG directed against either F or HN. Alternatively, agglutinated cells were treated with low concentrations of the above proteins (inducing only minor inhibition of either mobility or fusion), and immobilization of F and/or HN was induced by cross-linking with a secondary antibody; this protocol ensured a minimal contribution of direct binding to the viral proteins to the inhibition of fusion. Our results demonstrate that lateral immobilization of either F or HN results in a strong inhibition of cell-cell fusion and a much weaker inhibition of virus-cell fusion. The level of cell-cell fusion was directly correlated with the level of laterally mobile viral glycoproteins in the cell membrane (either F or HN). We conclude that lateral mobility of both F and HN in the red cell membrane is essential for cell-cell fusion and that not only F but also HN has a role in this fusion event. The possible reasons for the different dependence of cell-cell and virus-cell fusion on viral glycoprotein mobility are discussed.

In order to investigate the requirement for lateral mobilization of viral envelope glycoproteins on the cell surface in the induction of cell-cell fusion, we employed fluorescence photobleaching recovery to study the effect of the fusion temperature on the lateral mobilization of Sendai virus glycoproteins in the human erythrocyte membrane. As the fusion temperature was reduced below 37 degrees C (to 31 or 25 degrees C), the rates of virus-cell fusion, the accompanying hemolysis, and cell-cell fusion were all slowed down. However, the plateau (final level) after the completion of fusion was significantly reduced at lower fusion temperatures only in the case of cell-cell fusion, despite the rather similar final levels of virus-cell fusion. A concomitant decrease as a function of the fusion temperature was observed in the fraction of cell-associated viral glycoproteins that became laterally mobile in the erythrocyte membrane during fusion, and a strict correlation was found between the level of laterally mobile viral glycoproteins in the cell membrane and the final extent of cell-cell fusion. The accompanying reduction in the lateral diffusion coefficients (D) of the viral glycoproteins (1.4-fold at 31 degrees C and 1.9-fold at 25 degrees C, as compared to 37 degrees C) does not appear to determine the final level of cell-cell fusion, since fusing the cells with a higher amount of virions at 25 degrees C increased the final level of cell-cell fusion while D remained constant.(ABSTRACT TRUNCATED AT 250 WORDS)

We have recently developed a method to quantitate the fusion of reconstituted viral envelopes with cells by fluorescence photobleaching recovery (FPR) (Aroeti, B & Henis, Y I, Biochemistry 25 (1986) 4588). The method is based on the incorporation of non quenching concentrations of the fluorescent lipid probe N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)phosphatidylethanolamine during the reconstitution of the viral envelopes (the latter probe does not incorporate efficiently into the membrane of native virions). In the present work, we employed the fluorescent dye octadecyl rhodamine B chloride (R18), which can be incorporated directly into the membrane of native enveloped virions, to extend the FPR method to study fusion between native Sendai virions and intact human erythrocytes. The R18 fluorescence was found to be quenched in the viral envelope at the concentration range required for the FPR experiments, possibly due to preferential insertion of the probe into specific domains in the viral membrane. We therefore developed a correction (presented in the Appendix) which takes into account the lower quantum yield of the probe molecules in the membranes of unfused virions in the calculation of the fraction of fused virions from the FPR experiments. The results demonstrate that the method does indeed measure virus-cell fusion, and that the contribution of exchange to the measurements is not significant. The applicability of the method was further verified by the similarity of the results to those obtained independently by fluorescence dequenching measurements, and its ability to measure the distribution of virus-cell fusion within the cell population was demonstrated. These results suggest that the use of R18 can enlarge the scope of the FPR experiments to study the fusion of native virions with cells.

A method to quantitate viral envelope-cell fusion at the single-cell level is presented. The method is based on the incorporation of nonquenching concentrations of a fluorescent lipid probe into the viral envelope; fluorescence photobleaching recovery (FPR) is then applied to measure the lateral mobilization of the probe in the cell membrane following fusion. In adsorbed (unfused) viral envelopes, the probe is constricted to the envelope and is laterally immobile on the micrometer scale of FPR. After fusion, the envelope lipids intermix with the plasma membrane, the probe becomes laterally mobile, and the fraction of fused viral envelopes can be extracted from the fraction of mobile probe molecules. The method has several advantages: (i) It clearly distinguishes fused from internalized envelopes, as probes in the latter are immobile in FPR studies; (ii) focusing the laser beam on specific regions of the cell enables region-specific measurements of the fusion level; (iii) one cell is measured at a time, enabling studies on the distribution of the fusion level within the cell population. The new method was employed to study fusion of reconstituted Sendai virus envelopes (RSVE) containing N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine with several cell types. Experiments with human erythrocytes demonstrated that the lateral mobilization measured is due to fusion and not the result of exchange processes. The extent of RSVE-erythrocyte fusion determined by FPR was similar to that measured by two other independent methods (fluorescence dequenching and removal of adsorbed RSVE by dithiothreitol).(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of Sendai virus glycoproteins with cell membranes was proposed to increase the lateral mobility of membrane proteins, enabling membrane fusion and the aggregation of intramembrane particles by thermotropic separation (Volsky, DJ & Loyter, A, Biochim biophys acta 514 (1978) 213 [13]; Maeda, T et al. Exp cell res 123 (1979) 333 [15]; and Kim, J & Okada, Y, Exp cell res 132 (1981) 125 [44]). In order to test this hypothesis, we employed fluorescence photobleaching recovery to investigate the effects of Sendai virus-induced fusion on the lateral mobility of membrane proteins and lipids in a variety of cell types (human erythrocytes, BHK21, HeLa, 3T3 NIH, and mouse spleen lymphocytes). The results of the lateral diffusion measurements demonstrate that no significant alterations occur in the lateral motion of membrane proteins or a fluorescent phospholipid on all the cell types examined, including cells which revealed high susceptibility to the virally mediated fusion (human erythrocytes and BHK21 cells). These findings suggest that a permanent increase in the lateral mobility of cell surface components does not generally occur during Sendai virus-induced cell fusion, and thus cannot play a role in the fusion mechanism. The possible involvement of transient alterations in the lateral mobility of membrane components in the fusion mechanism is discussed.