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Vol. 11, Issue 11, 3765-3775, November 2000
and
*Department of Molecular Biophysics and Physiology, Rush Medical
College, Chicago, Illinois 60612; and A hemagglutinin (HA) of influenza virus having a single
semiconserved Gly residue within the transmembrane domain mutated to
Leu (G520L) was expressed on cells; these cells were bound to red blood
cells. By decreasing pH at 23°C rather than 37°C, an intermediate
with properties expected of hemifusion just as the membranes are about
to transit to full fusion was captured. As evidence: 1) increasing
temperature to 37°C at neutral pH allowed fusion to proceed; 2) after
achieving the intermediate, the two membranes did not separate from
each other after proteolytic cleavage of G520L because cells treated
with proteinase K could not fuse upon temperature increase but could
fuse upon the addition of chlorpromazine; and 3) at the point of the
intermediate, adding exogenous lipids known to promote or inhibit the
creation of hemifusion did not significantly alter the lipid dye spread
that occurred upon increasing temperature, implying that at the
intermediate, contacting membrane leaflets had already merged. A stable
intermediate of hemifusion that could transit to fusion was also
generated for wild-type HA, but pH had to be reduced at the
significantly lower temperature of 4°C. The fusion pores generated by
G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained
via a point mutation without the need for unusually low temperature
supports the hypothesis that hemifusion occurs before pore formation.
Department of
Biochemistry, University of Texas Southwestern Medical Center at
Dallas, Dallas, Texas 73235
Corresponding author. E-mail address:
fcohen{at}rush.edu.
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