The Transmembrane Domain (TMD)AS a Candidate for the Voltage Sensor in Fusion Mediated by Class II and Class III Viral Fusion Proteins

Abstract

We have shown that fusion of cells expressing class II or III proteins and fusion of pseudovirions bearing these proteins to target cells is voltage dependent: fusion is inhibited by positive trans-membrane voltages across the target membrane. In contrast, fusion mediated by class I viral proteins is independent of voltage. We showed that voltage dependence is shallow, ∼e-fold for 25 mV changes and that the voltage-dependent steps are downstream of hemifusion. These findings would be consistent with TMDs as the voltage sensor. We tested this by preparing chimeras consisting of the ectodomain of influenza HA (a class I protein), the TMD of VSV G (a class III protein), and the cytoplasmic tail of either influenza HA (yielding HGH) or VSV G (HGG). The chimeras and WT HA were separately expressed in effector cells and fusion to target cells was monitored by aqueous dye spread. Fusion required that the effector cells be pretreated with trypsin (to cleave the HA0 ectodomains) and low pH. Voltage was made positive across cell membranes by addition of a sodium ionophore (SQI-Pr). In the absence of ionophore (trans-negative voltage), fusion proceeded for WT HA and the two chimeras. But the addition of the ionophore abolished fusion mediated by either of the chimeras. This strongly indicates that the TMD of VSV G confers voltage sensitivity to the fusion process. Supported by NIH GM27367.