Voltage dependence is determined by the identity of the TMD.

<p>A. Cell-cell fusion induced by WT-HA was independent of voltage (first columns). But swapping the TMD of VSV G for that of HA resulted in fusion that was much greater for negative (filled bars) voltages than for positive (open bars) voltages (generated by the addition of SQI-Pr), independent of whether the CT was that of VSV G (second columns) or HA (third columns). Error bars are SEM (n = 3). B. Electrical capacitance measurements show that fusion pores did not form at positive voltages across the target membrane when a chimera, containing a TMD derived from VSV G, was the fusion protein. The initial pore conductances and early patterns of growth (at negative voltages) do not depend on the identity of the TMD. The conductance profiles are not markedly different from those of WT influenza HA (top trace) when both the TMD and CT of VSV G are swapped for those of HA (middle traces), or just the TMD (bottom traces) is switched. The electrical capacitance measurements confirm the conclusion from ionophore experiments that the dependence of fusion on voltage follows the class of viral protein from which the TMD is derived. C. Reducing the length of the TMD of a class I fusion protein does not confer voltage dependence to fusion. The CT of the fusion protein of JRSV Env was eliminated and the TMD truncated one amino acid at a time. Truncation reduced the extents of fusion compared to WT, but fusion remained independent of voltage for all lengths of the TMD. The horizontal axis denotes the last (C-terminal) residue of each TMD truncation. Error bars are SEM (n ≥3).