• Virology 253, 65–76 (1999)

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    • Abstract: Virology 253, 65–76 (1999)Article ID viro.1998.9493, available online at http://www.idealibrary.com onAn Analysis of the Role of the Target Membrane on the Gp64-induced Fusion PoreIlya Plonsky,* Myoung-Soon Cho,* Antonius G. P. Oomens,† Gary Blissard,† and Joshua Zimmerberg*,1

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Virology 253, 65–76 (1999)
Article ID viro.1998.9493, available online at http://www.idealibrary.com on
An Analysis of the Role of the Target Membrane on the Gp64-induced Fusion Pore
Ilya Plonsky,* Myoung-Soon Cho,* Antonius G. P. Oomens,† Gary Blissard,† and Joshua Zimmerberg*,1
*Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, Maryland 20892–1855; and †Boyce Thompson Institute, Cornell University, Tower Road, Ithaca, New York 14853–1801
Received June 5, 1998; returned to author for revision July 22, 1998; accepted October 27, 1998
Influenza hemagglutinin (HA) and GP64 of the baculovirus Autographa californica multicapsid nuclear polyhedrosis virus
induce strikingly different initial fusion pores when mediating fusion between host cells that express these fusion proteins
and target cells (Plonsky and Zimmerberg, 1996; Spruce et al., 1989, 1991; Zimmerberg et al., 1994). However, in these
experiments, variations in host and target membranes confounded the analysis of the role that major components of the
fusion reaction play in determining initial pore characteristics. To determine the contribution of the target cell plasma
membrane to the fusion pore phenotype, we studied GP64-induced fusion of stably transfected cells (Sf9Op1D) to either red
blood cells (RBCs) or Sf9 cells. Initial fusion pores in Sf9Op1D/RBC and Sf9Op1D/Sf9 cell pairs exhibited the same conductance,
and pore flickering was not observed in either combination of cells. This indicates that the target cell determines neither the
size nor the reversibility of the initial pore. However, the target cell does influence the kinetics of pore formation. The waiting
time between triggering and pore appearance was shorter for Sf9Op1D/RBC fusion than for Sf9Op1D/Sf9 pairs. No correlation
between pore waiting time and conductance was found. This argues against a molecular model that assumes aggregation
of the pore wall from a nonfixed number of components as the rate-limiting step in GP64 fusion pore formation. © 1999
Academic Press
INTRODUCTION 1- to 3-ms time resolution, have a small initial conduc-
tance of 0.6 nS (Spruce et al., 1989, 1991; Zimmerberg
With the introduction of admittance measurements in
et al., 1994). Initial fusion pores induced by the GP64 of
single-cell electrophysiology (Neher and Marty, 1982),
the Autographa californica multicapsid nuclear polyhe-
more detailed investigations of the key processes of
drosis virus (AcMNPV) have a higher conductance ( 1.3
membrane fusion have become possible. An analysis of
nS) than those mediated by HA, do not flicker, and ap-
fusion-induced changes in cell admittance has shown
pear 0.6 s after triggering (Plonsky and Zimmerberg,
that biological fusion occurs through a small pore linking
1996). Because studies on GP64- and HA-mediated
two previously separated aqueous compartments (Zim-
pores were conducted in experimental systems with dif-
merberg et al., 1987; Breckenridge and Almers, 1987).
ferent host and target cells and protein surface densities,
The conductance and kinetics of the formation and wid-
it is unclear to what extent these differences in the
ening of the fusion pore yield information about the
properties of pores induced by these fusion proteins are
immediate mechanisms of fusion, such as the molecular
due to variations in their sequences, their expression
size of the initial fusion site, the reversibility of its forma-
levels, or variations in the composition and energetics of
tion, and the driving forces behind the subsequent fusion
the membranes that they fuse.
pore widening.
Structure–function relationships between fusion pro-
Although viral envelope proteins are the best charac-
teins and fusion pores will probably become as impor-
terized fusion proteins, electrophysiological analyses of
tant for investigations into the mechanism of fusion as
their fusogenic properties in cell membranes are limited
they are now for the study of ionic channels. However, for
(Lanzrein et al., 1993; Plonsky and Zimmerberg, 1996;
proper experimentation and analysis, a defined system
Spruce et al., 1989, 1991; Tse et al., 1993; Zimmerberg et
for expression, membrane fusion, and electrophysiolog-
al., 1994). Large variability in the characteristics of fusion
ical recordings should be developed. To date, only HA-
pores has been reported. Fusion pores mediated by
induced fusion pores have been studied using the same
influenza hemagglutinin (HA) appear 30 s after trigger-
host cells (HAb2) and different target membranes (RBC
ing by low pH, sometimes flicker (open and close before
and planar lipid bilayers; Melikyan et al., 1995a, 1995b;
the final irreversible opening), and, when measured with
Spruce et al., 1989, 1991; Zimmerberg et al., 1994). Dif-
ferent conductance and tendencies to flicker were re-
1
To whom reprint requests should be addressed at Building 10,
ported in each of these studies, suggesting that the
Room 10D14, 10 Center Drive, MSC 1855. Fax: (301) 594-0813. E-mail: target membrane greatly affects the process of fusion.
[email protected] However, for HAb2/bilayer fusion, the target membrane
0042-6822/99 $30.00
65 Copyright © 1999 by Academic Press
All rights of reproduction in any form reserved.
66 PLONSKY ET AL.
has considerable surface tension (minimal in cells), cells, we generated and selected a stably transfected
which pulls open the fusion pore (Chizmadzhev et al., cell line that expresses GP64Op on the cell surface at
1995; Melikyan et al., 1995b). Thus the role of different levels similar to those of infected cells. To generate cell
target cell membranes in determining the initial fusion lines constitutively expressing the GP64Op protein, Sf9
pore phenotype remains unknown. cells were cotransfected with plasmid p64–166 (Blissard
Data on natural variability in fusion protein sequence and Wenz, 1992), which encodes the OpMNPV gp64
between different species of viruses accompanied by open reading frame under the control of an optimized
observations of dissimilarities of their pore phenotypes OpMNPV early promoter construct (Blissard and Rohr-
could provide insights into structure–function relation- mann, 1991), and with plasmid pAcie1-Neo, which en-
ships, particularly when modeling mechanisms of fusion. codes a bacterial neomycin resistance gene under the
The GP64 protein of another species, Orgyia pseudo- control of the AcMNPV ie1 promoter (Monsma et al.,
tsugata multicapsid nuclear polyhedrosis virus (OpMNPV), 1996). After selection in medium containing G148, sev-
efficiently mediates membrane fusion and is capable of eral cell lines expressing high levels of GP64 were iden-
functionally complementing a deletion of the gp64 gene tified by immunofluorescence microscopy (not shown)
in the AcMNPV genome (Monsma et al., 1996). The and flow cytometry. One cell line, Sf9Op1D, was selected
ectodomains of OpMNPV and AcMNPV fusion proteins for use in the current study because it demonstrated the
(GP64Op and GP64Ac, respectively) have 80% identical following combination of characteristics. Sf9Op1D ex-
amino acid sequences, with a conserved hydrophobic pressed high GP64 surface levels, with a relatively nar-
fusion domain and a large leucine zipper motif. Although row variation in surface levels of GP64 among cells. We
most differences in the ectodomains of these two pro- compared cell surface GP64 expression (Fig. 1) from cell
teins represent conservative substitutions, some of lines Sf9Op1D and Sf9Op64–6 (a previously generated cell
these substitutions are located within and adjacent to line expressing GP64Op; Monsma et al., 1996), AcMNPV-
the previously identified hydrophobic membrane fusion infected Sf9 cells (24 h postinfection), and transiently
domain (Blissard and Rohrmann, 1989; Monsma and transfected Sf9 cells expressing GP64Op (48 h posttrans-
Blissard, 1995) and thus could affect the characteristics fection). In previous studies, it was demonstrated that
of the fusion pore. cell line Sf9Op64–6 expresses similar surface levels of
To study the role of the target membrane in determin- GP64 to those found on the surface of AcMNPV-infected
ing the fusion pore phenotype, we developed a defined Sf9 cells (Monsma et al., 1996). However, the cell-to-cell
cell system for fusion protein expression, membrane variation in surface densities of GP64 in Sf9OP64–6 cells
fusion, and electrophysiological recordings by generat- was somewhat higher than that of AcMNPV-infected Sf9
ing a stably transfected GP64Op-expressing cell line, cells, as shown in Figs. 1B and 1D. This wide variation
Sf9Op1D. Sf9Op1D cells were used as the hosts in all was also observed in transiently transfected cells ex-
experiments, whereas the targets were either wild-type pressing GP64 (Fig. 1E). In contrast, the cell line gener-
Sf9 cells or RBCs. We found that the conductance of the ated for the current study, Sf9Op1D, exhibits surface levels
initial fusion pore induced by GP64Op does not depend and the more narrow cell-to-cell variation in surface
on the target cell, and the pore does not flicker in either expression of GP64 that more closely approximate those
combination of cells. This suggests little influence of the found in AcMNPV-infected Sf9 cells (Figs. 1B and 1C).
target membrane on initial pore characteristics. How- Abundant cell surface localization of GP64Op was also
ever, the pore waiting times (interval between triggering confirmed by immunofluorescence microscopy (Fig. 2).
and pore formation) measured for Sf9Op1D/RBC pairs
were significantly shorter than those for Sf9Op1D/Sf9
Binding of RBCs to insect cell membrane
cells, indicating that the target membrane can affect the
kinetics of fusion pore formation. For fusion pores, mea- The transfer of RBCs to the experimental chamber
sured in the Sf9Op1D/RBC system, there was no correla- induced binding of erythrocytes to a fraction of Sf9Op1D
tion between their initial conductance and waiting times. cells. Some Sf9Op1D cells bound few (up to 10) RBCs.
This argues against a model in which aggregation of a These aggregates were observed in transmission elec-
nonfixed number of fusion protein trimers is assumed to tron microscopy (Fig. 3A). Micrographs with high magni-
be a rate-limiting step in fusion kinetics. fication (Fig. 3A, inset) showed that the distance between
the two bound membranes is comparable to the thick-
RESULTS ness of the membrane ( 10 nm), matching the height of
the baculovirus spikes believed to be built of GP64 (Ad-
Stably transfected cells expressing OpMNPV GP64
ams et al., 1977). Binding of AcMNPV baculovirus to Sf9
Because previous studies have shown wide variation cells was recently characterized (Wang et al., 1997). To
in protein expression from cells transiently transfected examine whether binding of RBC to Sf9Op1D cells was
with plasmids expressing the GP64 protein (Monsma et induced by GP64, RBCs were added to wild-type Sf9
al., 1996) and because OpMNPV does not replicate in Sf9 cells. To our surprise, Sf9 cells were also able to form
TARGET MEMBRANE ON THE Gp64-INDUCED FUSION PORE 67
FIG. 2. Immunolocalization of OpMNPV GP64 on the surface of stably
transfected cells. Control Sf9 cells (A and B) or stably transfected
Sf9Op1D cells (C–F) were fixed, immunostained with an anti-GP64 Mab
(AcV5), and examined by immunofluorescence microscopy. The GP64
protein was detected in the cytoplasm and at the surface of Sf9Op1D
cells that were fixed and permeabilized with methanol (C and D). When
Sf9Op1D cells were fixed with paraformaldehyde (not permeabilized),
GP64 was detected only at the cell surface (E and F). No immunostain-
ing was detected on control Sf9 cells fixed with methanol (A and B).
Confocal immunofluorescence micrographs (A, C, and E) represent
1- m optical sections, and light micrographs represent differential
interference contrast images (B, D, and F).
close contacts with RBC (Fig. 3B). However, the contact
area between Sf9Op1D cells and RBCs appeared to be
more extensive than that between Sf9 and RBCs. Al-
FIG. 1. Analysis of surface-localized GP64 on individual cells by flow though more quantitative study is required to fully inves-
cytometry. Cells were incubated with an anti-GP64sol polyclonal serum tigate this observed binding phenomena, the simplest
in the presence of sodium azide and washed, and relative GP64 levels explanation is that Sf9Op1D cells can form specific (GP64-
were measured with an FITC-conjugated secondary antibody. Cell related) as well as nonspecific contacts with RBCs.
counts are plotted on the y axis and relative fluorescence levels on the
x axis. For each cell population examined, control cells were incubated
with only the FITC-conjugated secondary antibody (solid black line). To
Admittance analysis of GP64-induced fusion
measure relative levels of surface GP64, cells were incubated with both Fast delivery of the acidic solution triggered the
primary and secondary antibodies (dotted line). For reference, the
vertical dashed line on the left represents the mean fluorescence
transfer of membrane dye from bound RBCs to Sf9Op1D
intensity obtained in negative control experiments (A), in which unin- cells, signifying the pH-dependent development of a
fected Sf9 cells were incubated with anti-GP64 primary antibody and hydrophobic pathway between two cells (Fig. 4). In 35
FITC-conjugated secondary antibody. The vertical dashed line on the of 53 experiments, acidification caused characteristic
right in B and C represents the mean fluorescence intensity for GP64 changes in the pipet–cell admittance: a reversible
detected from Sf9 cells infected with AcMNPV (B). (C–E) Relative levels
of GP64 detected from two stably transfected cell lines (Sf9Op1D and
alteration in the real component with a peak value of a
Sf9Op64–6) and transiently transfected cells (Sf9, transfected), respec- few nS, an increase in the imaginary component to a
tively. maximum value of 10 nS (Fig. 5A), and a current
68 PLONSKY ET AL.
FIG. 3. Binding of RBC to insect cells observed by transmission electron microscopy. Low magnification micrographs A and B show Sf9Op1D (A)
and wild-type Sf9 cells (B) with bound erythrocytes (bar equals 1 m). Contact areas between Sf9Op1D and RBC and the wild-type Sf9 cell and RBC
are shown with high magnification in the corresponding insets (bar equals 100 nm).
discharge seen in cell conductance (Fig. 5B, inset 1). between Sf9Op1D and erythrocytes developed through
These changes in Y as well as GDC were induced by the characteristic stages of initial pore appearance,
membrane fusion (Breckenridge and Almers, 1987; Ne- followed by pore widening, stabilization of the inter-
her and Marty, 1982; Zimmerberg et al., 1987). In cellular conductance between a few and tens of nS,
agreement with previous findings (Spruce et al., 1989, further pore dilation, or a jump to an even higher
1991), RBC capacitance had a mean value of 1.0 0.2 conductance (Fig. 5A, inset). These stages were de-
pF, as calculated according to Eq. 2. Fusion pores scribed for mast cell exocytosis (Curran et al., 1993),
TARGET MEMBRANE ON THE Gp64-INDUCED FUSION PORE 69
one that was characteristic of SF9Op1D/RBC fusion (Fig.
5B, inset 2). All these differences in the electrophysio-
logical properties were the result of the higher mem-
brane capacitance of Sf9 cells (10 3.8 vs 1 0.2 pF for
Sf9 cell and RBCs, respectively). To prove this, computer
simulations of pore widening were performed as follows.
Theoretically, the fusion-induced increment of Re equals
( Cm2)2/[Gp(1 ( Cm2/Gp)2)], and an increment of Im
equals Cm2/[1 ( Cm2/Gp)2] (Zimmerberg et al., 1987).
When Sf9 cell or RBC Cm2 mean values were substituted
in the above expressions and Gp was increased up to 1
S, the calculated values of Re and Im were consistent
with those observed in experiments. Also, the target
membrane capacitance, calculated from the current tran-
sient time constants and known initial Gp, matched the
corresponding values of Cm2 found by admittance mea-
surements (Fig. 5B, insets 1 and 2). This indicates that
the duration of current transients in Sf9Op1D/RBC and
Sf9Op1D/Sf9 cells reflected the difference in membrane
capacitance of Sf9 cells and RBCs.
The conductance of the initial fusion pore
For Sf9Op1D/RBC pairs, the mean conductance of the
initial fusion pore was 1.2 0.7 nS (range, 0.3–2.6 nS;
n 33). Two pores in this group exceeded 4 nS. We
assumed that the transition of these pores into the wid-
ening stage was faster than our time resolution and have
not included them in further statistical analysis. In the
case of Sf9Op1D/Sf9 pairs, the mean initial Gp was 1.4
0.7 nS, varying between 0.2 and 2.8 nS (n 21). The
histogram of the initial Gp for each combination of cells
is shown in Fig. 5B. Because both samples passed the
test for normality of distribution and had equal variance,
two means were compared using an unpaired t test,
which showed no statistically significant difference be-
tween these two samples of conductance. To check for
the minimal detectable difference in sample mean val-
FIG. 4. Fusion of Sf9Op1D with human erythrocytes monitored by the ues, the power of the t test was calculated. The results of
dye redistribution technique. Fusion was triggered by a pressure pulse
calculations indicated that given these sample statistics
applied to a delivery pipet filled with acidic solution. RBCs were labeled
with a membrane dye PKH26. Cells are shown in bright-field (A) and and sizes, we could detect a significant difference be-
fluorescent microscopy before (B) and 3 min after (C) triggering. tween the two means if Gp in Sf9Op1D/RBC pairs was
0.8 nS.
Flickering (opening and closure of the pore before
as well as for HA-induced fusion of fibroblasts with
irreversible widening) was observed in neither Sf9Op1D/
artificial membranes (Melikyan et al., 1993).
RBC nor Sf9Op1D/Sf9 cell pairs. Similar pores are also
A similar progressive stage-like development of the
characteristic of GP64Ac-induced fusion: their mean con-
fusion pore conductance was observed after acidifica-
ductance is 1.3 0.6 nS and they never close (Plonsky
tion of Sf9Op1D/Sf9 cell pairs in 21 of 32 experiments.
and Zimmerberg, 1996).
Typically, in Sf9Op1D/Sf9 pairs, Re (real part of a pipet–cell
admittance) reached its maximum after a few tens of nS
Kinetics of fusion pore formation
and did not completely return to the baseline level as in
Sf9Op1D/RBC fusion. Im (imaginary part of a pipet–cell For Sf9Op1D/RBC fusion, waiting times between acidi-
admittance) increased to a higher value of 100 nS (not fication and initial pore formation varied between 0.164
shown). A transient change in GDC (due to the charging and 4.650 s, with a median of 0.440 s. Sf9OP1D/Sf9 cell
of the membrane of the nonclamped target Sf9 cell to the fusion exhibited a longer tw: the median value was 0.883
holding potential of the host cell) lasted longer than the s (range, 0.270–5.340 s). Because both samples of data
FIG. 5. GP64Op-induced Sf9Op1D/RBC cell fusion, studied by the time-resolved admittance measurement technique. (A) Fusion-related alterations
in both real and imaginary components of the cell admittance. Changes in Re, Im were triggered by a short pressure pulse (between arrows) applied
to the delivery pipet, which contained acid solution. Direct current conductance (GDC) showed no drastic change during fusion. Fusion pore
conductance for the time interval indicated by the bar was calculated and plotted in the inset. Here, the arrow indicates initial fusion pore appearance.
Further pore widening was accompanied by increasing noise due to vanishing Re (see Eq. 1). (B) The distribution of the initial pore conductance
for Sf9Op1D/RBC (black columns) and Sf9Op1D/Sf9 (white columns) cell–cell fusion. Initial fusion pores (F) in Sf9Op1D/RBC (inset 1) and Sf9Op1D/Sf9
(inset 2) pairs are shown with accompanied current discharges (E). In the recordings displayed in the insets, background holding currents have been
subtracted. Point-to-point intervals equaled 1 ms. Notice that the initial pore shown in inset 2 was stable, despite its large conductance. For the
Sf9Op1D/Sf9 pair, a current discharge lasted longer due to the higher value of the target cell capacitance. Current discharges were fit to the
single-exponential decay function I I0 exp( t/ ), where the time constant, , equals Cm2/Gp (insets 1 and 2). Values of were 1.04 ms for RBCs
and 6.8 ms for Sf9 cells. The capacitance, calculated from and Gp, was 1.04 pF for RBCs and 10.2 pF for Sf9 cells, matching the values of Cm2 for
both types of cells using admittance measurements.
TARGET MEMBRANE ON THE Gp64-INDUCED FUSION PORE 71
FIG. 6. Distributions of waiting times for Sf9Op1D/RBC (circles) and Sf9Op1D/Sf9 (triangles) pairs were plotted as survival probability points, Pt, as
explained under Materials and Methods. Pt were fit to the theoretical probability function, P(t), derived on the assumption of the multielement parallel
model (Eq. 3). The resulting curve for fusion of Sf9Op1D/RBC pairs is shown as a solid line. Inset, waiting times between acid solution delivery and
fusion pore formation in Sf9Op1D/RBC cell pairs were plotted against corresponding initial pore conductance. The Pearson correlation coefficient, rs,
shows no significant association between these two variables.
failed to pass the normality test, nonparametric statistics to 19 monomers (6 2 trimers) of GP64 to provide
were used for their comparison. The Kolmogorov-Smir- sufficient structural elements to form a fusion-competent
nov (KS) test showed that the difference between these complex. For Sf9Op1D/Sf9 fusion, the fitting algorithm did
two median values was statistically significant (Dmax not converge to a minimum value of 2; thus no unique
0.38, Dtab 0.37). set of parameters was obtained.
Experimental waiting times, prepared as a semilog To determine the strength of the association between
survival plot, were fit to Eq. 3, derived from a multiple the initial pore conductance and waiting time values, we
parallel element model (MPM) (Plonsky and Zimmerberg, used the Pearson correlation test (Fig. 6, inset). The
1996), in which fusion is controlled by independent ele- value of P and rs obtained for Sf9Op1D/RBC fusion (0.750
ments a and b, and for fusion to occur, n independent and 0.006, respectively) shows that there is no correla-
elements a and one element b should all enter their tion between initial Gp and tw, signifying that the forma-
permissive state (see Materials and Methods for details). tion of larger initial pores does not require longer (or
Results of the fit are shown in Fig. 6. The following shorter) waiting times.
parameters and the coefficient of the determination were
obtained (given with standard error): n 19.9 6.6, k1 DISCUSSION
12.7 1.5 s 1, k 1 0.3 0.1 s 1, k2 2.3 0.1 s 1
(R2 0.994). For Sf9Op1D/RBC fusion, the n value was the In the present study, we characterized the fusion pore
same as that obtained for fusion of Sf9 cells infected with formed by the OpMNPV baculovirus envelope glycopro-
AcMNPV baculovirus (Plonsky and Zimmerberg, 1996). tein GP64Op. The fusion protein was expressed in the
Assuming that the a element of the model corresponds stably transfected cell line Sf9Op1D. Sf9Op1D cells were
to a fusion peptide or cytoplasmic domain, of which there mixed with different targets: erythrocytes or wild-type Sf9
is thought to be only one per monomer, one can specu- cells. Fusion pores measured in Sf9Op1D/RBC pairs have
late that the results of our fit for n 19 would correspond the same conductance as those characteristic of
72 PLONSKY ET AL.
Sf9Op1D/Sf9 cell fusion, and flickering of the pore was not strongly indicate a predominant role of the target mem-
observed in either combination of cells. This indicates brane in determining the initial pore phenotype. The
that the target cell neither determines the size of the calculated power of the performed t test showed that if
initial pore nor changes the irreversible character of the the initial Gp in Sf9Op1D/RBC pairs is the same as that
pore. However, the target cell influences the kinetics of characteristic for HA (0.6 nS), it would have been de-
the fusion pore formation. Waiting times for Sf9Op1D/RBC tected in our experiments. Thus the assumption that the
pairs were shorter than those for Sf9Op1D/Sf9 cells. No initial conductance of the fusion pore in Sf9Op1D/RBC
correlation between the fusion pore waiting time and pairs is the same as that characteristic of HA-induced
conductance was found, indicating that the formation of cell/RBC fusion can be safely rejected.
larger initial pores does not require longer or shorter In the present study, we found that in a well-defined
waiting times. cellular system in which all major components partici-
pating in fusion except the target membrane remain the
The target membrane determines neither the same, neither the initial conductance nor the reversibility
conductance nor the reversibility of the fusion pore of the initial pore formation is dictated by the target
membrane: GP64Op-induced pores between Sf9Op1D/
Earlier, the architecture of the GP64-induced initial RBC cells or Sf9Op1D/Sf9 cells were identical. We con-
fusion pore was visualized as entirely made of bent clude that the conductance and flickering of the initial
bilayers enclosed within a proteinaceous fusion complex fusion pore are determined either by the host membrane
formed by a ring of GP64 trimers (Plonsky and Zimmer- or by the fusion proteins in the fusion aggregate. The
berg, 1996). The lipidic nature of the initial fusion pore influence of the palmitatoylated cystein residues in the
adopted for the GP64 fusion “machine” is essential to a cytoplasmic tail of HA on fusion pore flickering was
class of models (Chernomordik et al., 1995; 1998; Nana- recently revealed (Melikyan et al., 1997), supporting the
vati et al., 1992; Zimmerberg et al., 1991) that are strongly involvement of fusion proteins in flickering.
supported by the data showing that (1) both wild-type
and a mutation of influenza HA lead to hemifusion (Cher- Fusion proteins that belong to two different
nomordik et al., 1998; Kemble et al., 1994; Melikyan et al., baculoviruses form similar fusion pores
1995c) and (2) the inhibition and promotion of fusion
correlate with lipid intrinsic curvature (Chernomordik et Fusion pores between two AcMNPV virus-infected Sf9
al., 1995). cells were recently studied (Plonsky and Zimmerberg,
Theoretically, lipid composition can determine pore 1996). GP64Ac-induced pores have characteristics simi-
reversibility (flickering). If the intrinsic radii of curvatures lar to those mediated by GP64Op: their initial conduc-
of contributing species are energetically unfavorable tance exceeds 1.0 nS, and they form rapidly and never
(have negative curvature), the resulting force in the most flicker. A comparison of pores induced by two closely
bent region of the pore could cause pore closure (Kozlov related proteins in AcMNPV-infected Sf9 cells and
et al., 1989; Nanavati et al., 1992). Dimpling of the target Sf9Op1D/Sf9 pairs showed no statistically significant dif-
membrane toward the host membrane inside fusion pro- ferences in their initial conductance; any difference ex-
tein walls was suggested by models of exocytotic ceeding 0.5 nS would be detected. The difference in the
(Monck and Fernandez, 1992) as well as viral fusion median waiting time in infected cells (0.6 s) and Sf9Op1D/
Sf9 pairs (0.9 s) was not significant, as tested by non-
“machines” (Blumenthal et al., 1995). Therefore, the target
parametric statistics. Although the power of the t test
membrane might line the narrowest, conductance-limit-
was not sufficiently high to detect small differences in
ing part of the initial fusion complex and influence pore
pore conductance, we conclude that the divergence in
size and reversibility. To date, differences in experimen-
GP64Ac and GP64Op sequence was not reflected in the
tal systems (i.e., variations in fusion proteins, and in host
characteristics of the initial fusion pore.
and target membranes) have confounded the analysis of
the role that major components of the fusion reaction
Kinetic evidence argues against rate-limiting
play in determining initial pore characteristics. Therefore,
assembly of the fusion site from an arbitrary
it remains unclear whether the observed differences
number of GP64 trimers
between pores induced by GP64 and HA are due to the
sequence and stoichiometry of fusion proteins or due to The aggregation of a pore complex from a number of
the composition and energetics of membranes.


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