Fusion activity and inactivation of influenza virus: kinetics of low pH-induced fusion with cultured cells

Abstract

The kinetics of fusion of influenza virus (A/PR/8/34) with human promyelocytic leukaemia (HL-60), human T lymphocytic leukaemia (CEM) and murine lymphoma ($49) cells were investigated. Fusion was demonstrated by electron microscopy, and monitored by fluorescence dequenching of octadecylrhodamine incorporated in the virus membrane. Rapid fusion was induced upon mild acidification of the medium. At pH 5, all virus particles were capable of fusing with the cells. The initial rate and the extent of fusion were maximal between pH 4-9 and 5.2 and declined sharply below and above this range. The rate constants of adhesion of influenza virus to cells or erythrocyte ghosts were large, indicating a diffusion-controlled process. The rate constants of fusion of the virus with cells were smaller than those found previously for fusion with various liposomes. Although preincubation of the virus at acidic pH in the absence of target membranes almost completely inactivated the virus in its ability to fuse with erythrocyte ghosts, it reduced the extent of fusion with cultured cells by only 20 to 40%. Kinetic analysis of fusion revealed a mode of inactivation of the virus bound to erythrocyte ghosts or suspension cells, below pH 5-4, different from that of the virus preincubated at low pH without target membranes.

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

“…This result is particularly interesting in view of the fact that the rate of low pH inactivation is dramatically reduced upon lowering the temperature (3,13,14,20). Truly, a situation can arise where the enhanced rate of low pH inactivation at higher temperatures can result in a reduction in the final extents of fusion.…”

“…At pH 5 and 37°C, influenza virus exhibits 100% fusion activity toward the acidic liposomes PS and cardiolipin, whereas only 20 -40% of the virions are capable of fusing with liposomes composed of PC/PE with or without cholesterol and gangliosides (2,4). The phenomenon of partial fusion activity should not be confused with low pH inactivation of fusion activity that has been observed in the case of influenza virus (3,4,(13)(14)(15)(17)(18)(19)(20)(21). Thus, the reduction in the fusion rate constant upon exposure of influenza virus to low pH is similar when the target membrane of a liposome is composed of PS or PC/PE/cholesterol with or without gangliosides, but the final extents of fusion are 100% and 20 -40%, respectively (4).…”

Interactions of Influenza Virus with Cultured Cells: Detailed Kinetic Modeling of Binding and Endocytosis

“…The procedure has yielded the values for the rate constants of the aggregation and fusion processes, during virus-cell interaction, thus enabling Nir et al to obtain a more detailed description and prediction of the kinetics of the overall fusion process (21). This model has also been employed to evaluate the kinetics of fusion between influenza virus and liposomes (23), of Sendai virus fusion with phospholipid vesicles, erythrocyte ghosts, and cells (11,21,22), and of influenza virus fusion with cells lacking an endocytic capacity (3,18). By employing a similar formalism, we provided equations for uptake of particles (liposomes) by cells via endocytosis (20,24).…”

Construction and Characterization of Insect Cell-Derived Influenza VLP: Cell Binding, Fusion, and EGFP Incorporation

“…The fusion process was monitored by fluorescence dequenching of R18 due to the lateral diffusion and dilution from viral to cellular membrane as a result of membrane fusion [29, 30]. 6 × 10 6 of Jurkat cells pretreated with 25 mM of NH 4 Cl at 37°C for 15 min were suspended in 500  μ l of PBS containing 25 mM of NH 4 Cl and added 10  μ l of R18-labeled VLPs (protein estimation 4.7  μ g) and then incubated at 37°C for 10 min.…”