The Role Of Class Ii And Iii Viral Protein Transmembrane Domains (tmds) In Voltage-dependent Fusion

Abstract

We have found that fusion mediated by class II (e.g. Semliki Forest Virus E1) and class III (e.g., Vesicular Stomatitis Virus G) viral proteins are promoted by a trans-negative voltage and prevented by a trans-positive potential across the target membrane. Endosomes, from which these viruses normally fuse, naturally have a trans-negative potential and so voltage dependence may have important biological relevance. We have shown that in contrast to class II and III proteins, fusion induced by class I viral proteins (influenza HA, HIV Env, and ASLV Env) is independent of the target membrane voltage. We are currently seeking to identify the voltage sensor. Because the sensor must lie within the membrane to respond to the electric field, the fusion peptides and TMDs are sensor candidates. We therefore prepared viral protein chimeras that consist of the class I protein influenza HA as ectodomain, and the TMD and/or the cytoplasmic tail (CT) of VSV G. All chimeras fuse well to red blood cells, in accord with previous demonstrations that the precise TMD and CT of HA were of relatively little importance in fusion to red cells. But surprisingly, all chimeras were less efficient that WT HA in fusing to nucleated cells. We have therefore produced fluorescently-labeled pseudovirus using WT and chimeras as the envelope protein and are now monitoring fusion of these pseudovirions to voltage-clamped target cells. Supported by Public Health Service grants R01 GM27367 and GM057454.