Patch clamp studies of single cell-fusion events mediated by a viral fusion protein

Abstract

To enter cells, viruses must fuse their envelope with a host cell membrane. Fusion is mediated by specific, membrane-spanning fusion proteins, of which the influenza virus haemagglutinins (HA) are the best characterized. Several HAs have been sequenced, and the crystal structure of the major part of one HA is known. The conditions for fusion and some of the rearrangements in the HA that accompany fusion are well understood, but it remains unclear how HA causes bilayers to fuse. We have observed, in real time, unitary cell-fusion events caused by HA. Fibroblasts expressing HA were induced to fuse with red blood cells by a rapid drop in pH. Fusion was monitored by fluorescence microscopy, and by measuring the membrane conductance and capacitance of the fibroblast. The earliest event observed was the sudden opening of an aqueous pore connecting the cytoplasms of the fusing cells. Initially, the pore conductance often fluctuated between zero and approximately 600 pS, as if the pore were opening and closing repeatedly. Later, it increased over tens of seconds, as if the pore dilated. We suggest that, as in exocytosis, HA-mediated membrane fusion begins with the formation of a narrow pore. Based on the conductance, we estimate the initial diameter of the pore to be no more than twice that of a gap junction channel.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

“…The growth of pores over time also depended on the combination of the TM domain and CT present. WT HA-induced fusion pores often shut and then quickly reopened (Figure 8), a phenomenon known as "flicker" (Spruce et al, 1989;Zimmerberg et al, 1994). WT HA ( Figure 9B, open circles) and HPH-induced pores ( Figure 9B, triangles with cross) did not grow over 20 s ( Figure 9B) and in fact did not enlarge significantly over a 2-to 3-min period (our unpublished results).…”

“…As pores enlarged readily for HPP* cells, we could be reasonably certain that the full capacitance of each fused RBC was electrically revealed. The total increase in HA cell membrane capacitance should be approximately equal to the product of the number of current transients and the average RBC membrane capacitance of ϳ1 pF (Spruce et al, 1989). This was the case.…”

“…Sample sizes: WT HA (open circles), n ϭ 32; HPP* (filled triangles), n ϭ 20; HPstop (open triangles), n ϭ 28; and HPH (triangles with cross), n ϭ 27. The approximate diameters of fusion pores in nanometers (Spruce et al, 1989) are shown on the right axis. (A) The initial conductance of pores induced by HPP* was statistically (p Ͻ 0.001) larger than formed by the other constructs.…”

“…The same solution but buffered with 20 mM Cs-succinate, pH 4.8, was used to trigger fusion. Patch pipettes were filled with (in mM) 155 Cs glutamate, 5 MgCl 2 , 5 bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, and 10 Cs-HEPES buffer, pH 7.4 (Spruce et al, 1989;Spruce et al, 1991). Fusion was triggered by ejecting the pH 4.8 solution from another micropipette placed ϳ70 m from the cell.…”

Structures of vesicular stomatitis virus glycoprotein: membrane fusion revisited

“…The next step would be the formation of a pore in the fusion diaphragm. The initial pore is small and is often opening and closing repeatedly (the so-called flickering pore) before its enlargement that leads to complete fusion [7].…”