Voltage Across the Target Cell Membrane is a Strong Regulator of Fusion of Virus Containing Class II or Class III Proteins

Abstract

Simultaneous electrophysiological voltage clamp and confocal microscopy were used to investigate the dependence of fusion on the voltage across the membrane of the target cell for class II and class III fusion proteins. Cells expressing the fusion protein of Semliki Forest Virus or Venezuelan Equine Encephalitis Virus, both class II, or Vesicular Stomatitis Virus, class III, fused to target cells that were voltage-clamped to a trans-negative potential. Fusion did not occur for positive potentials. This pattern also held for fusion of pseudotyped virions to cells. These virions contained one of the three types of fusion proteins and a fluorescent lipid in the envelopes, and GFP within their core, allowing both hemifusion and fusion to be monitored after acidification of the external medium. Fusion of the pseudovirions exhibited the same voltage-dependence as found for cell-cell fusion. Hemifusion occurred for either voltage polarity at physiological temperature, but the kinetics of hemifusion depended on polarity and lipid dye transfer from virus to cell was aborted for positive, but not for negative potentials. The latter strongly indicates that hemifused membranes separate back into two distinct membranes for positive potentials, accounting for the block of fusion for this polarity. Creating hemifusion at low temperature allowed the hemifusion stage to be captured and studied. Fluorescent lipid dye clearly left the virus and content mixing was subsequently observed for trans-negative potentials, showing that hemifusion is a functional intermediate of fusion in this system. Voltage may be an important driving force for infection by all viruses that utilize class II and III proteins. Supported by NIH GM 27367.