Lateral mobility of reconstituted Sendai virus envelope glycoproteins on human erythrocytes: correlation with cell-cell fusion

We have recently employed fluorescence photobleaching recovery (FPR) to demonstrate that the envelope glycoproteins of Sendai virions become laterally mobile on the surface of human erythrocytes following fusion [Henis, Y. I., Gutman, O., & Loyter, A. (1985) Exp. Cell Res. 160, 514-526]. In order to investigate whether this lateral mobilization is involved in the mechanism of virally mediated cell-cell fusion, or is merely a result of viral envelope-cell fusion, we have now performed FPR studies on erythrocytes fused with reconstituted Sendai virus envelopes (RSVE). These RSVE, which were prepared by solubilization of Sendai virions with Triton X-100 followed by removal of the detergent through adsorption to SM-2 Bio-beads, fused with human erythrocytes as efficiently as native virions but induced cell-cell fusion to a much lower degree. The fraction of the viral envelope glycoproteins that became laterally mobile in the erythrocyte membrane following fusion was markedly lower in the case of RSVE than in the case of native virions. The lower cell-cell fusion activity of the RSVE does not appear to be due to inactivation of the viral fusion protein, since the envelope-cell fusion and hemolytic activities of the RSVE were similar to those of native virions. Moreover, fusion with RSVE or with native virions resulted in the incorporation of rather similar amounts of viral glycoproteins into the cell membrane. Since the reduced fraction of laterally mobile viral glycoproteins correlates with the lower cell-cell fusion activity of the RSVE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion.

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.     

Effects of fusion temperature on the lateral mobility of Sendai virus glycoproteins in erythrocyte membranes and on cell fusion indicate that glycoprotein mobilization is required for cell fusion

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)     

The lateral mobility of cell membrane components is not altered following cell fusion induced by Sendai virus

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.     

Rotational mobility of Sendai virus glycoproteins in membranes of fused human erythrocytes and in the envelopes of cell-bound virions

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)     

Alteration of membrane conductivity and fluidity in human erythrocyte membranes and erythrocyte ghosts following gamma-irradiation

Alterations of electrical properties of human erythrocyte membranes induced by gamma irradiation have been studied by means of conductivity measurements in the frequency range from 10 KHz to 100 MHz. The results clearly demonstrate the role played by haemoglobin in the structural modification of the membrane produced by gamma irradiation. Further support for this point of view has been derived from electron spin resonance measurements carried out on the same samples, labelled with different spin labels which probe the outer half layer of membrane at different penetration levels.     

Unmasking of a potassium leak in resealed human red blood cell ghosts.

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0 degrees C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

The spontaneous activation of a potassium channel during the preparation of resealed human erythrocyte ghosts

Resealed erythrocyte ghosts prepared under conditions which deplete the cell of its endogenous chelators and metabolites are found to be selectively permeable to potassium. The net efflux of potassium is stimulated by low concentrations of external potassium and can be inhibited by oligomycin. The effect is not expressed when resealed ghosts are formed by hemolysis in the presence of chelators or magnesium. The spontaneously activated pathway is actually the calcium-activated potassium channel, first discovered by Gardos in 1958. In the intact cell, the combined actions of the calcium pump and endogenous chelators maintain the calcium concentration below the threshold for activation. Current observations indicate that the channel is spontaneously activated by traces of calcium originating from the cell itself or from the unavoidable background of calcium found in the media. The channel in ghosts depleted of endogenous chelators exhibits its high affinity for calcium. Channel activation occurs during hemolysis and persists throughout subsequent washings.     

Fusion of Sendai virions with phosphatidylcholine-cholesterol liposomes reflects the viral activity required for fusion with biological membranes

Sendai virus envelopes were reconstituted after solubilization of intact virions with either Triton X-100 or octylglucoside. Envelopes obtained from Triton X-100, but not from octylglucoside solubilized virions, were hemolytic and promoted cell-cell fusion. Fluorescence dequenching studies [using N-4-nitrobenzo-2-oxa-1,3-diazole phosphatidylethanolamine-bearing viral envelopes] revealed that both preparations fused with negatively charged phospholipids. Fusion with phosphatidylcholine (PC)/cholesterol (chol) liposomes was promoted only by the hemolytic viral envelopes. Fluorescence dequenching studies, using intact virions bearing octadecylrhodamine B chloride, revealed that intact virions fused with PC/chol as well as with negatively charged phospholipids. Only fusion with PC/chol liposomes was inhibited by phenylmethylsulfonyl fluoride and dithiothreitol, reagents which are known to block the viral ability to fuse with biological membranes.     

Viral and non-viral induced fusion of pronase-digested human erythrocyte ghosts.

Human erythrocyte ghosts but was able to fuse only iso-human erythrocyte ghosts. Iso- and hypo-human erythrocyte ghosts were incubated with the proteolytic enzyme pronase under isotonic (iso-human erythrocyte ghosts) or hypotonic (hypo-human erythrocyte ghosts) conditions. Gel electrophoresis and electron microscope (freeze-etching) studies revealed that most of the erythrocyte membrane polypeptides were hydrolyzed by pronase under hypotonic conditions. Sendai virus readily agglutinated both pronase-digested iso-human erythrocyte ghosts and hypo-human erythrocyte ghosts were fused by the non-viral fusogenic agent glyceromonooleate. Freeze-etching studies revealed that during fusion the membranes of pronase-digested human erythrocyte ghosts are intermixed.     

Reconstitution of the basal calcium transport in resealed human red blood cell ghosts

The (45)Ca(2+) influx into right-side-out resealed ghosts (RG) prepared from human red blood cells (RBC) was measured. The (45)Ca(2+) equilibration occurred with t(1/2)=2.5 min and the steady-state was reached after 17 min with the level of 22+/-2 micromol/L(packed cells) at 37 degrees C. The rate of the influx was 97+/-17 micromol/L(packed cells)h. The (45)Ca(2+) influx was saturated with [Ca(2+)](0) at 4 mmol/L and was optimal at pH 6.5 and 30 degrees C. Divalent cations (10(-4)-10(-6)mol/L), nifedipine (10(-5)-10(-4)mol/L), DIDS (up to 10(-4)mol/L), and quinidine (10(-4)-10(-3)mol/L), inhibited the (45)Ca(2+) influx while uncoupler (10(-6)-10(-5)mol/L) stimulated it. In contrast to intact RBC, vanadate inhibited the (45)Ca(2+) influx when added to the external medium, however, the stimulation was observed when vanadate was present in media during both lysis and resealing. PMA had no effect under conditions found to stimulate the Ca(2+) influx in intact RBC. The results show that the Ca(2+) influx into RG is a carrier-mediated process but without control by protein kinase C and that the influx and efflux of Ca(2+) are coupled via the H(+) homeostasis similarly as in intact RBC but with modified mechanism.     

Sendai virus envelope glycoproteins become laterally mobile on the surface of human erythrocytes following fusion

Fluorescence photobleaching recovery has been employed to study the lateral mobility of the Sendai virus envelope glycoproteins (HN, neuraminidase/hemagglutinin protein (HN) fusion protein (F) on the surface of human erythrocytes. Our results indicate that the two viral glycoproteins are laterally immobile on the cell surface prior to fusion, and become mobile during the fusion process. The two fused glycoproteins are mobilized to the same extent (diffusion coefficients of 3.1-3.3 X 10(-10) cm2/sec with mobile fractions of 0.53-0.57 for both HN and F). Their mobilization is blocked under conditions that allow virus adsorption and hemagglutination, but not virus-cell or cell-cell fusion. These findings suggest a possible role for the lateral diffusion of the viral glycoproteins in the mechanism of cell-cell fusion, enabling them to perturb the membranes of adjacent cells and lead to cell-cell fusion.     

Unmasking of a potassium leak in resealed human red blood cell ghosts

A selective potassium leak is observed in resealed, human red blood cell ghosts when hemolysis is performed with distilled water at pH 6.5, 0° C. The leak, which has a maximum near pH 6.7, is suppressed when either magnesium or a chelating agent is present in the hemolysing medium. The potassium leak has the additional property that it can be suppressed after resealing by washing the ghost membranes in a medium containing a low concentration of ATP or EDTA. The data suggest that through the dilution of endogenous chelating agents at hemolysis a potassium leak may be unmasked.     

Lateral mobility of both envelope proteins (F and HN) of Sendai virus in the cell membrane is essential for cell-cell fusion

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.     

Cytoxicity of erythrocyte ghosts loaded with ribosome-inactivating proteins following fusion with CHO cells

The ribosome-inactivating proteins gelonin, Momordica charantia inhibitor, pokeweed antiviral protein, and one from Saponaria officinalis were enclosed in human erythrocyte ghosts. The proteins once trapped in ghosts and fused with CHO cells inhibited colony formation at concentrations of approximately 1 ng/ml (3 X 10(-11) M), whereas the free proteins only had an effect at concentrations of greater than 1 microgram/ml.