Partial Fusion Activity of Influenza Virus toward Liposomes and Erythrocyte Ghosts Is Distinct from Viral Inactivation

Abstract

Final extents of fusion of influenza virus (A/PR/8/34 strain) with neutral and partially acidic liposomes were monitored with (i) a fluorescence resonance energytransfer assay in which the liposomes were labeled and (ii) by the dequenching of octadecylrhodamine, initially incorporated in the viral membrane. The latter assay was also employed in the fusion of influenza virus and Sendai virus with erythrocyte ghosts. In all cases, a phenomenon of partial fusion activity of the virus was observed, which is distinct from low pH inactivation. The unfused influenza or Sendai virions, which were separated by sucrose gradient centrifugation from liposomes or erythrocyte ghosts exhibited again partial fusion activity toward freshly added liposomes or ghosts, respectively. The conclusion is that the fraction of initially bound and unfused virions does not consist of defective particles, but rather of particles bound to the target membranes via inactive sites on the virus (or on cellular membranes), or else, partial fusion activity is a manifestation of a certain probability of production of fusion inactive sites by irreversible association of viral glycoproteins or peptides in the target membrane.

Evidence that Rabies Virus Forms Different Kinds of Fusion Machines with Different pH Thresholds for Fusion

“…These results are reminiscent of results obtained for Sendai virus (28) and influenza virus (30) for which a phenomenon of partial fusion activity has also been described that was not due FIG. 4.…”

Tight Binding of Influenza Virus Hemagglutinin to Its Receptor Interferes with Fusion Pore Dilation

“…From these observations, we concluded that HA-receptor binding that is too tight interferes with the enlargement of fusion pores. Many studies have been published concerning the relationship between HA binding and fusion activities (1,7,12,13,15,20,21,23), and most indicate that the binding of HA to receptors is necessary for an efficient fusion reaction. The question is whether this concept is inconsistent with our conclusions from the present study.…”

Role of a transbilayer pH gradient in the membrane fusion activity of the influenza virus hemagglutinin: Use of the R18 assay to monitor membrane merging

“…Fusion was monitored continuously at pH 5.0 using the fluorescence dequenching assay as described previously (8-10). In brief, fusion of influenza virus with a target membrane will result in R18 dilution into the new bilayer, which, in turn, will promote a release in probe selfquenching, and a concomitant increase in the fluorescence of the sample.…”

“…For this purpose fusion was followed by the octadecylrhodamine B chloride (R18) dequenching assay (8-10), using liposomes as target membranes for intact virions. In order to simulate the outer monolayer of a target cell plasma membrane these model membranes were composed of the zwitterionic phospholipids PC and PE, and also included the sialic acid-containing ganglioside GD1a, a molecule known to act as a receptor for the virus (10, 11, 15, 20). Two populations of liposomes were prepared: one encapsulating an aqueous medium at pH 7.4, and one encapsulating exactly the same medium, but with its pH adjusted to 5.0.…”

“…Since it has been shown that liposomes are not always equivalent to biological membranes in the study of viral fusion activity (10), experiments using erythrocyte ghosts (resealed in medium buffered at either pH 7.4 or pH 5.0) as targets for influenza virus were also performed.…”