Translocation of a hydrocarbon fluorescent probe between Epstein-Barr virus and lymphoid cells: An assay for early events in viral infection

Abstract

Translocation of the hydrocarbon fluorescent probe diphenylhexatriene (DPH) between membranes was studied by fluorescence polarization (P) analysis. First, using a model system, the high P value (0.324) of DPH-labeled cholesterol/phosphatidylcholine liposomes and the low P value (0.157) of DPH-Iabele phosphatidylcholine liposomes allowed detection of DPH (6) is, in principle, an indirect measurement of the thermal mobility of the DPH molecules incorporated into a lipid bilayer. This is determined to a large extent by the lipid composition of the membrane (7). The degree of fluidity/rigidity of a membrane could be envisaged as a mechanical barrier imposed by the lipid bilayer on the degree of rotational mobility of DPH molecules (8). Therefore a slow rotation of DPH in a "rigid" membrane domain is recorded by a high P value, whereas a fast rotation of DPH in a "fluid" membrane region is measured by a low P value (6)(7)(8). Based on this assumption, fluorescence polarization analysis of DPH has been extensively used, in the last few years, to analyze the dynamic structural organization of lipids in cellular membranes (9-11) and viral envelopes (12,13). These studies have independently shown that the P values of DPHlabeled lymphoid cells are markedly lower (14, 15) than those of DPH-labeled enveloped viruses (12, 13). Moreover, it has also been shown that hydrophobic molecules such as cholesterol (16)(17)(18)(19) and DPH (20,21)

Interaction of polyunsaturated fatty acids with animal cells and enveloped viruses

“…Membrane rigidity is then determined by the protein-lipid interactions. In fact, the membrane "rigidity" of the viral envelope, which is determined by its lipid composition, is significantly higher than that of the host cell plasma membrane (5,6,10,12,16,18). These observations indicate that at the time of formation of the viral envelope the spatial organization of host cell lipids around the virus-specific proteins is selective (22).…”

Cell membrane potential changes follow epstein‐barr virus binding

“…EBV was grown in the B95-8 producer cell line and purified as described previously (Rosenthal et al, 1978). Each 100-p1 aliquot of virus suspension that was used contained virus concentrated from 0.5-1.0 liter of supernatant.…”

“…EBV, a herpesvirus, binds to a receptor found almost exclusively on B lymphocytes of humans and new world monkeys. These cells also express receptors for the third component of complement, C3b (Rosenthal et al, 1978;Yefenof, 1979;Yefenof et al, 1976). EBV infection of B cells results in stimulation of DNA synthesis and immortalization of the cells with little virus production.…”

The Herpesviruses

“…These results have been interpreted as indicating fusion of the more rigid viral envelope with the more fluid host-cell membrane. Since the half-time for dye transfer for both primary cells and Raji cells is the same, the mechanism for penetration for each type of cell is thought to be similar (Rosenthal et al, 1978).…”

“…Further evidence that EBV receptors are intimately related to C3 receptors is the observation showing that preincubation of cells with excess C3 blocks adsorption as measured by the transfer of dye and the reduction in the polarization of fluorescence (Rosenthal et al, 1978).…”