* Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183; Cellular Biophysics Laboratory,
National Institute of Bioscience and Human Technology, Tsukuba 305; and § Department of Biophysics, Faculty of Science,
Kyoto University, Kyoto 606, Japan
The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10-20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or "microprotrusions of the lipid bilayer." At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the "initial fusion pore" at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.
Viral membrane fusion is widely accepted as a paradigm for biological membrane fusion mechanisms
(Monck and Fernandez, 1992 HA is synthesized as a trimeric form of monomers with
an approximate molecular weight of 84,000. The precursory form of HA is converted to a fusion-active form by
posttranslational cleavage; each HA monomer is cleaved
into two polypeptide chains, HA1 and HA2 (Klenk et al.,
1975 In this study, we have investigated the structural features of membranes during fusion between influenza virus
and liposomes using EM. Since the process of viral fusion
with liposomes is completed very rapidly (within 1 min at
pH 5.0-5.2 and 37°C; Maeda et al., 1981 The following points are discussed. (a) Fusion intermediate: previous kinetic studies of HA-induced fusion demonstrated a delay time preceding the onset of fusion and
suggested the involvement of prefusion states (Morris et
al., 1989 (b) Structural features of the "initial fusion pore": Spruce
et al. (1989) (c) Multiple fusion sites on a single viral particle: we often observed that a single viral particle causes multiple fusion intermediates arranged in a polygon on the target liposome membranes. The fusion event seems to proceed
simultaneously on each fusion intermediate. We elucidated morphologically the sequence of the "single-point"
and "multi-point" fusion events.
(d) Structural changes in HA spikes under fusion-competent conditions: several lines of evidence (e.g., Harter et
al., 1989 (e) Molecular architecture of fusion intermediate: most
of the proposed models for HA-induced fusion intermediates have commonly postulated alignment of several HA
spikes closely surrounding the fusion site (Stegmann et al.,
1990 Materials
Influenza virus A/PR/8(H1N1) was grown in embryonated chicken eggs
and partially purified as described earlier (Maeda et al., 1975 Liposomes containing glycophorin were prepared essentially as described by MacDonald and MacDonald (1975) To characterize the interaction between the virus and the glycophorinbearing liposome quantitatively, binding and fusion of spin-labeled influenza virus with the liposomes was assayed essentially as described (Kawasaki and Ohnishi, 1992 Fig. 1 shows the effect of incorporation of glycophorin into liposomes
on viral binding and fusion. The virus bound to and fused even with receptor-free liposomes, although not efficiently, consistent with earlier studies
(Maeda et al., 1981
Samples for Quick-Freezing
(a) Influenza virus (total protein 80 µg) in 40 µl Pipes buffer was mixed
with 220 µg liposomes in 260 µl Pipes buffer. The mixture was kept for 15 min at 4°C and then centrifuged at 12,000 g for 5 min to obtain the pellet.
Membrane fusion was triggered at pH 5.0 and 23°C by mixing 15 µl of
the pellet with 3 µl of acidic buffer (80 mM Na-citrate, 70 mM NaCl, pH
adjusted to 4.6).
Three mixtures were incubated for 19 s, 30 s, and 2 min, respectively, at
23°C before quick-freezing. As a control at neutral pH , the pellet was suspended in Pipes buffer and incubated for 30 s at 23°C before being processed for quick-freezing.
(b) For the experiment using purified HA rosettes and liposomes, 200 µg HA rosettes was mixed in place of the virus using the protocol described in a and incubated for 30 s.
(c) Viruses incubated for 30 s at pH 5.0 and 23°C were processed by the
mica flake technique (Heuser, 1983 (d) Viruses alone incubated for 30 s at acidic and neutral pH, and liposomes alone incubated for 30 s at acidic pH were also processed for quickfreezing.
EM
The samples were quickly frozen using the technique of Heuser (1981).
The frozen materials were fractured and then etched for 2 min in a Balzers
BAF 300 (Balzers Union, Balzers, Liechtenstein) with the electron beam
gun mounted at 20° relative to the etched surface, to obtain replicas. The
replicas were observed with an electron microscope (model JEM 200 CX;
JEOL, Akishima, Japan) operating at 200 kV.
Shape of Spikes at Neutral and Acidic pH
Fig. 2 shows the appearance of viruses on replicas at neutral pH. The viruses were ~120 nm in diameter and were
homogeneously distributed when observed at low magnification (Fig. 2 a). The fracturing split the viral membrane
along its hydrophobic interior, resulting in two kinds of
fracture face (Fig. 2 b), the P-face and the E-face. Etching
revealed that the external surface of the viruses was covered with many spikes. The viral membrane is known to
have two kinds of spikes, HA and neuraminidase (NA). Although it appears difficult to clearly distinguish the two
types on a replica, the spikes which seem to have relatively
slender stalks connected to a P-face may be NA spikes
(Fig. 2 b, arrows). The molecular ratio of HA to NA has
been reported to be 5:1 (for review see Skehel et al., 1980
After the medium containing the viruses had been adjusted to pH 5.0, the samples were incubated for 30 s at
23°C, followed by replication (Fig. 3). Although fusion between viruses and liposomes should already have been under way by this time, the low pH had no detectable influence on the appearance of the HA spikes (Fig. 3, a and d).
No bending, tilting, opening, or lengthening was detected
in >90% of spikes on the replicas.
EM observations using negative staining by Ruigrok et
al. (1986) However, it was noteworthy that at low magnification
(Fig. 3 b), viruses in acidic media showed a tendency to aggregate with each other. Most of the HA spikes were ~14
nm long, whereas spikes at sites of contact between two viruses (Fig. 3 b, arrows) were apparently shorter than the
other spikes. In rare cases, short spikes were also observed
on solitary virions (Fig. 3 c, s), although it was not clear
whether the shortening was due to the acidification process itself or to freeze-fracturing.
Shape of Viruses on Mica Flakes
To obtain a closer view of the virus on replicas, we used
the mica flake technique. This involved mixing specimens
with an aqueous suspension of tiny flakes of mica, followed by quick-freezing for replication. However, this
technique significantly altered the shape of the viral particles incubated in medium at pH 5.0, as compared with
those incubated at pH 7.4. As shown in Fig. 4, b and c, at
pH 5.0 and 23°C, viruses were adsorbed on the surface of
the mica and appeared to be deformed either by some external force or because of the adsorption itself. Moreover,
observation of the fractured E-face suggested that the adsorption was so tight that the viral half membrane was as
flat as the mica surface. This distorted shape of the virus
particles suggested that they had struck the hard surface of
the mica so violently that the spikes in the center were
pushed back and everted to form projections on the E-face.
By contrast, on the mica surface at pH 7.4 (Fig. 4 a), the
viruses appeared to have landed very softly, leaving the
overall shape of the virus undisturbed. This could be seen
better in side-by-side comparisons; the viruses shown in
Fig. 4 c were fractured and etched at pH 5.0, whereas those
in Fig. 4 d were processed identically at pH 7.4. The fractured viruses on the mica after processing at pH 7.4 showed a natural curvature without everted spikes on the
E-faces, similar to those fractured in ice (see Figs. 2 and 3).
These results indicate that under the fusion-competent
condition (at pH 5.0 and 23°C, for 30 s) the overall structure of the HA spike does not alter, although the nature of
the spike is somehow changed so that it interacts strongly
with the surface of mica.
Viruses and Liposomes at Neutral pH
As a control for the "fusion experiments" under acidic
conditions, influenza virus and liposomes were mixed at
pH 7.4, and incubated for 30 s at 23°C. The liposomes were
composed of 80% egg phosphatidylcholine, 20% cholesterol, and 1% erythrocyte glycophorin, the viral receptor.
Although the liposomes contained the receptor, some of
the virions were not attached to the surface of the liposomes, as shown in Fig. 5, a and b. These free particles may
have been nonspecifically bound virions, which were released from liposomes between the centrifugation and
quick-freezing steps because of unstable binding (see Materials and Methods). However, the other virions were
shown to be located on the surface or in the vicinity of the
liposomes, and most of these may have corresponded to
virions that were bound specifically to the receptor. Even
at these binding sites, the fractured convex faces (Fig. 5, a
and d) and the fractured concave faces (Fig. 5 c) of the liposomes appeared very smooth and velvety and free from
any blisters or warts. It was not possible to observe glycophorin on fractured liposomal faces as IMPs or any other
microscopic structural feature. In addition, the liposomes alone at pH 5.0 showed the same velvety face as those at
pH 7.4 (see the concave faces shown in Fig. 10 g).
Viruses and Liposomes at Acidic pH
In contrast to the results obtained at pH 7.4, after only a
19-s incubation at pH 5.0 and 23°C, many viruses were adsorbed to the surface of the liposomes as if competing for
sites (Fig. 6 a). However, an extremely interesting finding
was the presence of blisterlike or wartlike small protrusions, each 10-20 nm in diameter, on the fractured convex
face of the liposomal membrane. These protrusions were
found alone or in triangular or rectangular arrangements.
However, the most interesting configuration formed by
the protrusions was a ringlike heptagon. These heptagons
often had an additional centrally located protrusion. In
Fig. 6 a, four heptagons are evident in the upper right region of the liposome face. Three of them have additional
protrusions in the center. The outer diameter of each heptagon was typically about 100 nm, roughly corresponding
to the diameter of the virus. The distance between the protrusions varied, but was usually ~40-50 nm. The area circumscribed by the heptagon on the face of the liposome appeared to show slight inward depression.
As shown in Fig. 6 b, the viruses were not of uniform
size, but both large and small ones coexisted in the same
sample. On the bottom of the concave fracture face of the
largest liposomal membrane, another heptagon arranged
in a ringlike configuration can be seen. This time, however, it is composed of seven pits on the vertices and a centrally located pit. Judging from the size of the heptagon, it
is undoubtedly complementary to the previous heptagon
composed of protrusions on the convex face. On closer observation, two virions can be seen directly below the polygonally arranged pits in Fig. 6 b (arrows). Judging by the
formation of these pits, it seems clear that the pits and protrusions on the faces of the liposomes are actually produced by the action of the virus and possibly by HA
spikes. We also observed that the area circumscribed by
the pit heptagon on the concave face appeared to be
pushed inward slightly. Some pits also existed solitarily on
the concave face.
Structures of Small Protrusions and Pits
Before discussing the role of the protrusion and the pit as
the "fusion apparatus," we must clarify their conformation
in membranes. For example, "inverted micelles" have
been reported on the fracture faces of artificial membranes (Cullis et al., 1979; for review see Verkleij, 1984 Since the small protrusions on convex faces and the
small pits on concave faces had the same polygonal arrangement, it is apparent that they result from the same
membrane domains and are complementary to each other.
In addition, the protrusions were observed only on convex
faces and the pits only on concave faces. These facts indicate that if the protrusions and pits are combined they reconstruct small bendings of the continuous lipid bilayer, which protrude outwardly.
Direct evidence for this interpretation is shown in the
large fractured face of a liposome in Fig. 8 b. The outer
line of the liposome, labeled M, represents the hydrophilic
surface of the outer leaflet of the unit membrane. The inner line labeled L delineates the hydrophobic interior of
the membrane, which at its closest point to the outer line
might roughly be considered to represent the outer leaflet
of the unit membrane. The outer and inner lines of the
convex liposome membrane are observed to run parallel
with each other in close apposition. They are parallel even
at the site of the projection labeled Pr, where the liposome membrane is fusing with a virus on the left.
Therefore, we consider that the protrusions on convex
faces and the pits on concave faces correspond to the
bends of a continuous lipid bilayer, and not to the lipidic
particles proposed for fusion between model membranes.
We refer to this structure as a microprotrusion of the lipid
bilayer or a microprotrusion of the membrane.
Further evidence for the structure of the microprotrusion is given in the section entitled Fusion between HAequipped Liposomes.
Fusion Events on Concave Faces
In Fig. 7a, a cluster of relatively small viruses is evident directly below a liposome with its concave face exposed. On
the concave face, just above the viruses, several solitary
pits can be seen. It is noteworthy that no pits are present
on the other parts of the concave face of this liposome. We
surmise that the solitary pits are also generated by the activity of viruses, mainly of smaller size.
Fig. 7 b shows the concave face of a liposome with pits in
various arrangements. Many are solitary, but some are
grouped rectangularly. Pentagonally arranged pits are also
present. Around the pit indicated by the arrow, the viral
and liposomal membranes show connections suggestive of
fusion between the two membranes.
Fig. 7 c is a high magnification micrograph of cross-fractured viral membranes and a liposome membrane. Many
slits or connections between the viral and liposomal membranes are clearly observable (arrows). The virus on the
far right may be connected to the liposome by two slits.
On a favorable fracture plane shown in Fig. 7 d, several
pits are observable on the concave face. However, one of
them appears to have been transformed into a narrow slit
bridging the liposome and the virus. When observed from
just above, as shown in Fig. 7 e (arrow), the slit was seen
actually to consist of a narrow channel ~4 nm wide. These
slits were thought to correspond to the initial fusion pore,
and would widen until omega-shaped fusion necks were
formed between the virus and liposome (Fig. 7, f and g).
Fusion Events on Convex Faces
On the convex face shown in Fig. 8 a, several solitary protrusions can be seen. Four viral membranes with exposed
E-faces adhere to the surface of the liposome in a shape
reminiscent of micropinocytosis. Fusion necks can also be
observed, apparently connected to solitary protrusions
(arrows).
A broad plane of the convex face of a liposome is shown
in Fig. 8 b. Here and there, protrusions arranged in heptagonal, triangular, or rectangular configurations are evident. Fusion rings can also be observed. There appears to
be one at the vertex of a triangle where a protrusion
should have been located (arrow labeled Po).
A polygonal ring composed of nine protrusions is shown
in Fig. 8 c. The diameter of each protrusion differs slightly.
The largest one indicated by an arrow has a tiny hole <4
nm in diameter in the center. A hole can also be seen in
the center of one protrusion which forms part of a triangular arrangement in Fig. 8 d (arrow). The diameter of this
hole is apparently larger than the others. Upon observing
the fusion rings associated with polygonally arranged protrusions on the convex faces shown in Fig. 8, e-g, we concluded that the fusion rings had formed from small holes
located in the centers of the microprotrusions.
Sequence of Fusion Events
Therefore we suggest that the sequence of events during
the process of fusion may be as follows: (a) A connection
between two membranes is made via microprotrusions
which may form from either side. (b) The initial fusion
pore, observed as a small hole and a narrow slit, is formed
in the center of each microprotrusion. (c) The initial fusion
pore expands to become visible as a larger fusion ring or a
larger fusion neck. (d) On a single virion, fusion may proceed in parallel on many of the microprotrusions that form polygons.
If this tentative hypothesis is true, we should be able to
observe the stages of the theoretical sequence of fusion
events depicted in the diagram shown in Fig. 9 a. We refer
to the sequence of fusion events depicted in the upper
model (Fig. 9 a, I) as single-point fusion, and that depicted
in the lower model (Fig. 9 a, II) as multi-point fusion. Relatively small virions appeared to undergo single-point fusion, as in Fig. 7, a, d, e, and f, whereas larger virions underwent multi-point fusion, as in Fig. 6 b. The sequence of
fusion events for single-point fusion has already been
shown in Fig. 7, d-g. This sequence could also be observed
in the case of multi-point fusion in a three-dimensional manner.
On the convex liposomal face in Fig. 9 b, many large
holes surrounding membrane protrusions are present. The
protrusion marked with an asterisk apparently corresponds to the stage marked with an asterisk in Fig. 9 a, as
seen from the outside. In the lower left corner of the concave face of the liposome in Fig. 9 c, several pits aligned on
a circle were seen. This appears to correspond to the asterisked stage in Fig. 9 a as observed from underneath. The
fracture shown here passed through the concave face of
the liposomal membrane and continued with the viral
membrane at the locations of the pits, then again continued with the liposomal membrane. If these pits were to
fuse and dilate, the central part of the membrane would
become isolated in the form of a vesicle, as shown in the
lower right corner of Fig. 9 a. In Fig. 9 d, two isolated vesicles can be observed in the fusion ring. Thus we surmise
from the morphological evidence that fusion actually occurs between the viral and liposomal membranes, using
tiny pits and protrusions as the fusion apparatus.
Nature of IMPs on Concave Faces
The concave fracture face of a liposome membrane fusing
with viral particles at 30 s after reduction of the pH of the
reaction mixture is shown in Fig. 10 a. Careful scrutiny reveals the presence of many pits on the plane. Surprisingly,
however, each pit is occupied by an IMP. Originally, the liposome membranes had no IMPs (Fig. 4). Therefore,
these particles present on the concave fracture face appear
to have issued from viral membranes as a consequence of
pH reduction.
Four pits are evident on the concave face of the liposome shown in Fig. 10 b. Three of them have no particles
in the center. However, the pit indicated by an arrow has a
very distinct IMP ensconced in the center. A virus is positioned directly below the pit containing the particle, suggesting that the particle itself could in fact be a part of HA
delivered from the virus. IMPs free from pits were also observed (arrowheads). The pits that did not contain IMPs
were larger than those that contained a particle.
Pits forming a polygon were also sometimes occupied by
IMPs, as shown in Fig. 10, c and d. In Fig. 10 e, there are
several IMPs at the neck of a membrane depression. Since
some IMPs on the concave face were free from pits, particles at the neck seemed to emerge from the pits and travel
outwardly along the interior of the liposomal membrane
away from the original polygonal area. Although IMPs were also located at the bottom of the depression, the particles at the neck were ~10 nm in diameter and larger than
those at the bottom. Measurement revealed that the smaller
particles were similar in size to the IMPs observed on the
viral E-face membranes.
To explore the nature of the IMPs on the fractured face
of liposomal membranes, we purified HA rosettes from intact influenza virus. The HA rosettes were mixed with liposomes at neutral pH, and incubated for 30 s at pH 5.0 and 23°C, followed by rapid freezing for replication. As
shown in Fig. 10 f, the HA rosettes were observed as aggregates of various sizes adhering to the outer hydrophilic surface of the liposome (arrows). No protrusions could be
found on the convex face, either solitarily or in the form of
polygons. Furthermore, no pits were evident on the concave face.
We examined the possibility that HA rosettes might
penetrate into the membrane interior to form IMPs. Observation of the reverse side of the fracture face confirmed
the existence of such IMPs, as shown in the inset of Fig. 10
f (arrows). The diameter of the particles was ~10 nm, resembling that of the larger IMPs ensconced in the pits. As
shown in Fig. 10 g, no IMPs were observed in the concave
faces of two liposomes frozen alone at pH 5.0. Therefore, there is good evidence to suggest that the particle ensconced in the pit is part of HA that has gained enough hydrophobicity at pH 5.0 to penetrate into the lipid membrane core.
Fusion between HA-equipped Liposomes
In the same samples in which we observed fusion events
between viruses and liposomes, some liposomes had already acquired HA spikes on their surfaces as a result of
fusion with viruses. The liposomes equipped with HA appeared to fuse with the same sequence of morphological
intermediates as that observed between viruses and liposomes. Perhaps it would be appropriate here to describe
briefly the observations we made concerning fusion between HA-equipped liposomes. We were able to obtain
some new findings due to the large radii of the two fusing
liposomes.
The etched surface of the liposome shown in Fig. 11 a
(30 s after acidification) appears very smooth, without any
incorporated material. However, the surface of the liposome in Fig. 11 b (same sample as that shown in Fig. 11 a)
has many projections, which would be HA spikes transferred from the viruses by fusion. A liposome densely covered by the projections is also shown in Fig. 11 c.
On convex liposomal faces (Fig. 11 d), protrusions were
also induced, but no regular heptagonal arrangement of
protrusions was observed, though a somewhat ringlike arrangement of larger size could be detected. Three fusing liposomes are shown in Fig. 11 e. Two of them, with concave
faces exposed, show a narrow channel <10 nm wide at the
fusion point (arrow). A low magnification view of two fusing liposomes is shown in Fig. 11 f. The protrusions, arranged in a ringlike manner on the convex face, adhere to
the covering liposomal concave membrane at the vertices
of the protrusions. Observed from the outside (Fig. 11 g),
the points of adhesion were bridges between the two fusing convex faces of the liposomes, which also clearly demonstrated the multi-point type of fusion shown in the asterisked drawing in Fig. 9 a. Fig. 11 h shows two fusing
liposomes with their concave faces exposed. In the center, two half membranes displaying a classical fused membrane-like junction have been fractured perpendicularly.
The junctional profile is associated with three pits, which
are paired with pits of equal size across the junction (arrows). If we imagine the complementary convex faces superimposed upon these concave faces, it becomes convincing that the connections between the two membranes are actually formed via membrane microprotrusions from
both sides.
On a favorable replica like that shown in Fig. 11 i, pits
and protrusions are seen to form a single polygon. Tiny
holes are evident in the center of the protrusions (short arrows). Around the hole indicated by a long arrow, two half
membranes, the upper concave and the lower convex, appear to be connected to each other. Two or three small pits
containing IMPs were observed on the concave face of the
upper liposomal membrane. These IMPs were presumed to have issued from the lower liposomal membrane. It
seemed most probable that there would be protrusions at
exactly the corresponding locations on the convex face directly underneath the IMPs. Also, the convex face complementary to the concave face bearing the pits would also
have protrusions at the sites directly above the pits. Thus
we conclude that a single IMP on one side of two fusing membranes can induce microprotrusions in both opposing
membranes.
In Fig. 11 j, we can see examples of events occurring
throughout the process of fusion between liposomes, exactly the same as those observed between viruses and liposomes. (a) A connection is made via microprotrusions
from both sides. (b) An initial fusion pore forms as a perforation in the center of each microprotrusion. (c) The
pores widen to form fusion rings. Thus in the case of fusion
between liposomes, the microprotrusions and the pits also constitute the fusion apparatus.
Asymmetrical Distribution of Protrusions, Pits,
and IMPs
In this study, we clarified the structure of microprotrusions
of the lipid bilayers between fusing influenza virus and liposomes, which were observed as small protrusions on the
fractured convex faces and as small pits on the concave
faces. Two previous studies have investigated HA-mediated fusion using quick-freezing replica techniques (Knoll
et al., 1988 Knoll et al. (1988) We think that our results are different from those of
Knoll et al., since they reported a symmetrical distribution
of small protrusions, pits, and large IMPs on the concave
and convex faces, whereas we found the small protrusions
exclusively on the convex face, and the pits and IMPs exclusively on the concave face.
An experiment similar to ours was performed previously by Burger et al. (1988) Contrary to the observation made by Burger et al., we
found that at neutral pH the virus did not induce any
structural change on the liposomal membrane; neither
protrusions on the convex faces nor invaginations on the
concave faces were observed. Therefore the 9-14-nm particle that Burger et al. observed at neutral pH on the liposomal convex face may be an entirely different structure from the microprotrusion we observed.
We found two kinds of IMPs on the concave face of liposomes processed at pH 5.0 with the virus. The smaller
IMPs, ~6 nm in diameter, are similar to those on the viral
E-face, which may correspond to the membranous segments of HA, and which are thought to be unrelated to the
fusion intermediate. On the other hand, the larger ones,
~10 nm in diameter, often appeared to be ensconced in
the pit, and it is noteworthy that the IMPs contained in the pits were positioned directly above the viruses bound to
the liposomes. Therefore we suggest that the larger particle may be a portion of the HA protein that includes the
fusion peptides, and may play a critical role in the fusion
event.
Fusion Pathway via the Microprotrusion of the
Lipid Bilayer
We concluded that the microprotrusion of the lipid bilayer
is the intermediary membrane structure that forms during
HA-induced fusion. A schematic drawing of the proposed
sequence of events is shown in Fig. 12 a. A drop in pH
causes the hydrophobic fusion peptide and some other
part of HA to penetrate into the lipid bilayer of the liposome, connecting strongly and bending the apposing membranes. The portion of HA that penetrates into the interior of the liposomal membrane is depicted as small solid
circles in the figure. When observed using the freeze-fracture technique, this part would appear as a small protrusion on a convex liposomal face, while on a concave face, it
would be seen as a pit containing an IMP (as shown in Fig.
12 a, I and I
As fusion proceeds, an aqueous pore must form between the viral membrane and the liposomal membrane. If
we observe the pore site on the convex liposomal fracture
face, a larger protrusion with a tiny hole in the center may
be seen, while on the concave face, we should see a pit of
small diameter containing no IMP (Fig. 12 a, II and II In the course of the expansion of fusion sites, fusion
pores may assume various diameters, as shown in the right
corner of Fig. 12 a. Since the fusion pores may form at
multiple sites on a single viral particle, growth of the fusion sites would produce an intervening membrane with a
complex morphology.
As we have described in Results, all the membrane
structures discussed here were actually demonstrated by
our EM observations.
Initial Fusion Pore
In patch-clamp studies of fusion between HAb-2 cells and
RBC, Spruce et al. (1989) Architecture of the Fusion Intermediate
A sectioned profile of the microprotrusion is shown schematically in Fig. 12 b. To our knowledge, no previous reports have described such a small membrane curvature as
that of the microprotrusion we observed (10-20 nm). It
should be noted that the thickness of a lipid bilayer is usually 4-5 nm. Because of the remarkably small diameter of
the microprotrusion, the packing of lipid molecules in the
top of the outer leaflet would be quite loose and fluid. By
contrast, the packing within the inner leaflet at the same site would be compressed and more rigid. We suggest that
this great difference in the packing of lipid molecules between the two lipid layers would be crucial for the merger
of the outer leaflets, and then the inner leaflets, of the two
membranes. In addition, the microprotrusion would require a rather small area of contact, minimizing the energy
needed to overcome the repulsive forces between the apposing membranes (Rand, 1981 Stegmann et al. (1990) However, with respect to the localization and morphology of spikes and IMPs, our observations are different
from the previous models. Although all of the schematic
drawings for the previous models include a fusion site
closely surrounded by several HA spikes, we observed neither spikes nor IMPs just around the protrusions and the
pits. Instead, we found that the large IMPs were ensconced in the centers of the pits. We suggest that the observed IMP might contain a portion of HA which penetrates into the liposomal membrane, since the IMPs were
observed on the array of HA spikes on the viral surface,
and also because isolated HA rosettes were observed as
similar large IMPs on the concave face of the liposome under acidic conditions. In this case, the size of the IMPs,
~10 nm, seems to be rather large for only three fusion
peptides. The IMP might thus consist of fusion peptides
along with some other elements of HA and lipid molecules.
In addition, the schematic models proposed by Stegmann et al. (1990) Therefore, it is possible to hypothesize another model
for the fusion intermediate (Fig. 12 d). The standing HA
spikes would tug and deform apposing membranes into
the shape of a microprotrusion if the spikes were centrally
located and deeply anchored to both of the membranes as
IMPs. Because we observed that fusion necks bore no
IMPs, we suggest that the IMP needs to move out from the microprotrusion by some unknown mechanism, since the
initial fusion pore is formed between the two membranes.
Recently, Carr and Kim (1993) reported that a synthetic
peptide corresponding to a "loop" region of the HA2 subunit form a coiled-coil upon acidification. They proposed a
"spring-loaded" mechanism, in which the conversion of
the loop to the coil extends the coiled-coil of the long
Our observation that the microprotrusion does not accompany bent spikes but bears a large IMP ensconced in
the center seems consistent with the spring-loaded mechanism. However, since we still know little about the structure of HA in the protein-lipid complex at the fusion site,
further studies are needed to clarify the relationship between the model schemes involving a spring-loaded mechanism, a fusion site surrounded by HA spikes, and a microprotrusion of the lipid bilayer with an IMP at its center.
In conclusion, we propose that the microprotrusion of
the lipid bilayer is a structure that represents a fusion intermediate. We believe that protein-mediated fusion events
must originate at the points where IMPs can be observed.
; White, 1992
). In
particular, hemagglutinin (HA)1 of influenza virus is the
best characterized among fusogenic membrane glycoproteins (for reviews see Hughson, 1995
; Carr and Kim, 1994
;
Brunner and Tsurudome, 1993
; Stegmann and Helenius, 1993
; Clague et al., 1993
; Wilschut and Bron, 1993
; Bentz
et al., 1993
; Wiley and Skehel, 1987
). HA possesses fusion
activity only at acidic pH (<pH 5.5), which is used for control of viral fusion with endosomal membranes in the
course of viral infection of host cells. The pH-dependent
conformational changes in HA and insertion of the "fusion peptide" of the protein into target membranes are considered to be the trigger that induces fusion.
; Lazarowitz and Choppin, 1975
; Maeda et al., 1981
;
for review, see Air and Laver, 1986
; White, 1990
). According to the three-dimensional structure of HA determined by x-ray crystallography (Wilson et al., 1981
), the trimeric
HA spike has a height of 135 Å from the surface of the viral membrane. The receptor binding site of HA1 is on the
top region of the spike, and the fusion peptide, ~20 amino
acid residues at the amino terminal of HA2 (Gething et
al., 1978
; Gething et al., 1986
), is located ~35 Å from the
viral membrane, >100 Å apart from the target membrane. In spite of the extensive characterization of this protein,
basic questions about the molecular mechanism of HAmediated membrane fusion, such as how HA causes close
apposition of two membranes and the type of fusion intermediate formed by HA, are still unanswered (Hughson et
al., 1995; Carr and Kim, 1994
).
; White et al., 1982
;
Stegmann et al., 1985
; Stegmann et al., 1990
; Kawasaki and
Ohnishi, 1992
), it is impossible to analyze the structural sequence of fusion events using conventional EM methods.
Although numerous biochemical and biophysical studies of virus and membrane fusion activity have been reported
(for reviews see Ohnishi, 1988
; Stegmann et al., 1989
;
White, 1990
; White, 1992
; Stegmann and Helenius, 1993
),
and several EM studies have been published (Matlin et al.,
1981
; Yoshimura et al., 1985
; Stegmann et al., 1990
), there
are only a few reports concerning HA-induced fusion
studied using quick-freezing replica techniques (Knoll et
al., 1988
; Burger et al., 1988
). To clarify the intermediary steps of membrane fusion, we took advantage of the deepetch replica technique developed by Heuser (1981).
; Sarkar et al., 1989
; Stegmann et al., 1990
; Ludwig
et al., 1995
). Several models for fusion intermediates have
been proposed (Stegmann et al., 1990
; Bentz et al., 1990
;
Guy et al., 1992
; Wilschut and Bron, 1993
; Bentz et al.,
1993
; Carr and Kim, 1994
). However, no direct morphological evidence for these models has been presented. In
this study, we show that HA induces "microprotrusions of
the lipid bilayer" in the precursory stage of fusion, and we
propose a fusion mechanism via this novel membrane
structure as an intermediate.
studied fusion between HA-expressing cells
(HAb-2) and RBC using fluorescence microscopy and
patch-clamp electrophysiology. They proposed that the
earliest event was the sudden opening of an aqueous pore
with a diameter of <4 nm between the two fusing cells (for
review see Monck and Fernandez, 1992
). Here we demonstrate, by direct EM observations, the formation of the initial fusion pore from the fusion intermediate and its dilation to accomplish the fusion event.
; Brunner, 1989
) suggest that the fusion peptide is
involved in the fusion event. The distance of >100 Å between the fusion peptide and the target membrane at neutral pH must be somehow shortened at acidic pH for the
peptide to be inserted into the lipid bilayer. To explain this
process, several models assuming drastic conformational changes in HA have been proposed (White and Wilson,
1987
; Stegmann et al., 1989
, 1990). In this study, however,
we did not detect these assumed drastic changes in the
spike structure on our quick-frozen replicas; no obvious
bending, tilting, or opening of spikes was observed under
fusion-competent conditions.
; Bentz et al., 1990
; Guy et al., 1992
; Bentz, 1993; Wilschut and Bron, 1993
). We discuss the molecular architecture of the fusion intermediate by examining the localization of intramembrane particles (IMPs) at the fusion sites.
Materials and Methods
). Virus stock
further purified by sucrose density gradient centrifugation was stored at
80°C until use. HA was purified from viral membrane in the form of rosettelike aggregates, as described previously (Kawasaki et al., 1983
). Viral
protein was assayed by Lowry's method (Lowry et al., 1951
) with BSA as
a standard. Egg yolk phosphatidylcholine was purified as described by
Singleton et al. (1965)
. The concentration of phospholipid was determined by phosphorus assay (Bartlett, 1959
). Cholesterol was purchased from
Wako Chemical Co. Ltd. (Osaka, Japan) and recrystallized before use.
Glycophorin was purified from human RBC membrane following the procedure of Segrest et al. (1979)
, and stored at
80°C.
. Aliquots of glycophorin in
10 µl water were added to 2 ml chloroform/methanol (2:1) containing a
lipid mixture (4 mg phosphatidylcholine and 1 mg cholesterol) and dried
in a test tube. Then 1 ml Pipes buffer (5 mM Pipes-NaOH, 145 mM NaCl,
pH adjusted to 7.4) was added with several glass beads, and the tube was
gently shaken for 15 min at 37°C. The resulting dispersion was centrifuged
for 5 min at 4°C to obtain the pellet, which was suspended in the original
volume of Pipes buffer.
). Spin-labeled virus (37 µg of total protein) in 50 µl Pipes buffer was mixed with 150 µg liposomes in 200 µl Pipes buffer.
The mixture was kept for 10 min at 4°C and then centrifuged at 12,000 g
for 5 min. The pellet was suspended in 20 µl of 20 mM Na-citrate, 130 mM NaCl, pH 5.0, and transferred to a quartz capillary tube at 4°C. Fusion efficiency (percent) after 5 min at 37°C was estimated from the increase in the
electron spin resonance (ESR) peak height. As an assay of viral binding to
liposomes, the amount of spin-label in the pellet was estimated from the
total intensity of the ESR signal and converted to the amount of viral total
protein.
, White et al., 1982
, Stegmann et al., 1986
, Kawasaki
and Ohnishi, 1992
). Inclusion of glycophorin into liposomes at a receptor/
lipid ratio exceeding 0.5% (by weight) increased the fusion efficiency from 17 to ~60%. The enhancement of viral fusion was receptor specific because the fusion efficiency for glycophorin-liposomes treated with
neuraminidase was almost the same as that for glycophorin-free liposomes
(data not shown). By contrast, enhancement of viral binding by glycophorin was much less marked. Although viral binding was increased twofold
by adding 1.0% glycophorin, it was below the saturation level. For the EM
experiments described below, we used liposomes with 1% glycophorin at
all times. The results shown in Fig. 1 confirm that the virus efficiently fuses
with liposomes containing 1% glycophorin. It is also evident that the pellet obtained from the virus and liposome mixture contains both specifically and nonspecifically bound virions.
Fig. 1.
Enhancement of binding (clear circles) and fusion
(solid circles) of spin-labeled influenza virus with liposomes by incorporation of glycophorin into the liposomes (see Materials and
Methods). For the binding assay, the amount of virus cosedimented with the liposomes was estimated from the total ESR signal. Because the virus bound even to liposomes without glycophorin, it is clear that the virus caused both receptor-specific
binding and nonspecific binding with the glycophorin liposomes.
Fusion efficiency was estimated from the increase of the ESR
peak height after 5 min at pH 5.0 and 37°C. Fusion was enhanced
by a much lower concentration of glycophorin than was the case
for binding.
[View Larger Version of this Image (17K GIF file)]
). As a control, viruses were also processed at neutral pH and 23°C for 30 s.
Results
).
Note the size of the IMPs. The particles on the E-face
were slightly smaller than those on the P-face.
Fig. 2.
Influenza viruses at pH 7.4 and 23°C. (a) Viruses 120 nm in diameter are distributed homogeneously. (b) A high magnification electron micrograph of two virions. Compare the size of IMPs on a P-face (Pf) to that of IMPs on an E-face (Ef). The measured diameter of IMPs on the E-face is ~6 nm. Arrows indicate what could be stalks of NA spikes. Bars, 100 nm.
[View Larger Version of this Image (141K GIF file)]
Fig. 3.
Influenza viruses incubated at pH 5.0 and 23°C for 30 s before freezing. (a and d) The appearance and the height of the spikes are unchanged from those at neutral pH. Arrows indicate stalked spikes grouped together. (b) Spikes at contact sites of virions (arrows) are apparently
shorter than the other spikes. (c) In rare cases, short spikes (indicated by s) are observed on solitary virions. Bars: (a, c, and d) 50 nm; (b) 100 nm.
[View Larger Version of this Image (163K GIF file)]
demonstrated that HA spikes on viral particles
(X-31 strain) and reconstituted vesicles were elongated by
1-2 nm on average after treatment at pH 5 for 10 min.
Since the resolution afforded by our EM technique with
replication was not as high as 1-2 nm, it was impossible for
us to detect this scale of elongation. Ruigrok et al. also indicated that in purified HA rosettes, the spikes are much
more elongated
to 21.1 nm on average
by low pH treatment. In addition, in aggregates of bromelain-released hemagglutinin induced by low pH treatment, the spikes were
elongated to 17.2 nm on average. However, our observations also did not reveal such extensive elongation of the
spikes. This discrepancy might have been due to the difference in the incubation time under low pH conditions; the
incubation conducted by Ruigrok et al. was for 10 min,
whereas our incubation was for 30 s. Alternatively, the discrepancy might have been due to the EM techniques used;
although they negatively stained their samples, we quickly
froze the samples without chemical fixation or staining.
Our finding that the overall structure of HA spikes does
not alter under fusion-competent conditions seems consistent with the cryo-EM observations reported by Booy (1993)
.
Fig. 4.
Appearance of frozen influenza viruses on the surface of mica. (a and d) Viruses incubated for 30 s at pH 7.4 and 23°C were mixed with mica flakes and frozen for replication. (b and c ) Viruses similarly processed at pH 5.0. For explanation see Results. Bars, 100 nm.
[View Larger Version of this Image (122K GIF file)]
Fig. 5.
Influenza viruses mixed with liposomes at pH 7.4 and 23°C, and incubated for 30 s before freezing. (a-d) Note that fractured convex faces (Vf) of the liposomes appear very smooth and velvety. Virions adhering to the surface of the liposomes are evident. Cf,
concave face of liposome. Bars: (a) 1 µm; (b-d) 100 nm.
[View Larger Version of this Image (167K GIF file)]
Fig. 10.
Influenza viruses
and liposomes (a-e), and HA
rosettes with liposomes 30 s
after acidification at pH 5.0 and 23°C (f and g). (a) Solitary pits on a concave liposome face. Some of them
contain IMPs. (b) The arrow
indicates a pit containing a
very distinct IMP ~10 nm in
diameter. Note that a virus
is positioned directly below
the pit. Arrowheads indicate
IMPs that are free from pits. Three larger pits contain no
IMPs. (c and d) Pits forming
a polygon are also occupied
by IMPs. (e) In terms of size,
there are two kinds of IMPs.
The IMP at the neck of the
depression is larger than the
one at the bottom. The measured diameter of the IMP at the bottom is about 6 nm. (f)
Arrows indicate HA rosettes
of various sizes on the outer
surface of the liposome. Note
that no protrusions are
present on the convex face.
(f, inset) Arrows indicate two IMPs ~10 nm in diameter on
the concave face of a liposome incubated with HA rosettes in acidic buffer for 30 s.
No pits can be seen on the
concave face. (g) Liposomes
alone frozen at pH 5.0. Although the magnification of
this figure is larger than that
of the inset in f, no IMPs can
be observed. Bars, 100 nm.
[View Larger Version of this Image (168K GIF file)]
Fig. 6.
Influenza viruses
and liposomes at 19 s after
acidification at pH 5.0 and
23°C. (a) Adsorption of viruses onto the liposome surface. The convex face of the
liposome membrane is exposed. Many small protrusions 10-20 nm in diameter
are evident. They are observed singly as well as in triangular, rectangular, and even pentagonal formations.
However, the most interesting configuration is a heptagon with an occasional centrally located protrusion. (b)
Two liposomes with their
concave fracture faces exposed. On the lower liposomal face a heptagon composed of seven small pits with a centrally located pit is
present. Judging from the diameter of the pit heptagon, it
is clear that it is complementary to the heptagon composed of seven small protrusions. Two large viruses are
observed just below the pits
arranged in a polygonal manner (arrows). These pits delineate the contour of viral
spikes. Some pits also exist
solitarily. Bars, 100 nm.
[View Larger Version of this Image (141K GIF file)]
).
These micelles present a structural appearance similar to
the small protrusions we observed on convex faces. Here,
we define a microprotrusion as having a bilayer arrangement of lipid molecules continuous with the rest of the
membrane.
Fig. 8.
Influenza viruses
and liposomes 19 s after acidification at pH 5.0 and 23°C. (a)
Several solitary protrusions
present on the convex face of a
liposome. Four viral E-face
membranes are adherent to
the surface of the liposome in a
shape reminiscent of micropinocytosis. Necks or "fusion
rings" apparently connected
with solitary protrusions (arrows) can also be observed. (b)
A variety of heptagonally, triangularly, or rectangularly arranged groups of small protrusions are evident on the broad
convex face of a liposome. The
arrow labeled Po indicates fusion rings apparently occupying the site of a protrusion in
the vertex of a triangle. For explanations of the arrows labeled M, L, and Pr, see Results. (c) Nine protrusions arranged in a polygonal manner. Note that each protrusion
differs slightly in diameter.
The arrow indicates the largest, which has a hole <4 nm in
diameter in the center. (d) A
hole is also evident in the center of a protrusion in a triangular group. Again, the hole is
found in the largest protrusion.
(e-g) Fusion rings associated with protrusions showing a polygonal arrangement. The rings
appear to have developed
from holes in the centers of the
protrusions. Bars, 100 nm.
[View Larger Version of this Image (163K GIF file)]
Fig. 7.
Influenza viruses and
liposomes 19 s after acidification at pH 5.0 and 23°C. (a)
Virions of smaller size are observed directly below a liposome
with its concave face exposed.
There are many solitary pits
just above the virions. Note
that no pits can be detected on
the remainder of the liposome
membrane. (b) Many solitary pits and a few rectangularly
grouped and even pentagonally grouped pits are present
on the concave face. Around
the pit indicated by an arrow,
the viral and liposomal membranes have connections suggestive of fusion between the
two. (c) Cross-fractured viral and liposome membranes. Arrows indicate slits between the
viral and liposomal membranes.
(d) Five solitary pits are present
on a concave face. One has
turned into a narrow slit bridging the liposome and a virus.
(e) Observed from just above
(arrow), the slit is seen to consist of a narrow channel ~4 nm
in diameter. (f and g) The slit
appears to widen until an
omega-shaped fusion neck is
formed. Bars, 100 nm.
[View Larger Version of this Image (170K GIF file)]
Fig. 9.
Influenza viruses and liposomes 30 s after acidification at pH 5.0 and 23°C. (a) A schematic drawing of two series of fusion events. We refer to the series in I as single-point fusion, and to that in II as multi-point fusion. (b) Many large holes are seen to surround
membrane protrusions on the convex liposomal face. The protrusion marked with an asterisk apparently corresponds to the asterisked
drawing in a. (c) A liposome with a concave face exposed is shown. Two or three solitary pits are present in the central part. On the left,
pits forming a circle are evident. This part corresponds to the labeled drawing as observed from underneath. (d) The convex face of a liposome. A fusion ring containing two vesicles is evident. These vesicles are apparently derived from the liposome membrane, as depicted in the lower right corner of a. Bars, 100 nm.
[View Larger Version of this Image (91K GIF file)]
Fig. 11.
Morphological sequences of fusion events
between HA-equipped liposomes in a virus-liposome
mixture 30 s after acidification at pH 5.0 and 23°C. For explanation see Results. Bars,
100 nm.
[View Larger Version of this Image (161K GIF file)]
Discussion
; Burger et al., 1988
). In these studies, particles,
pits, and invaginations were observed on target liposomal
membranes, which were apparently similar to those we observed.
studied the fusion of liposomes with
the plasma membrane of cultured cells infected with influenza virus. After a 30-s incubation at pH 5.0 and 37°C,
they observed well-defined IMPs and pits on the concave
and convex fracture faces of the liposomes and on those of
the plasma membrane. These particles were thought to be
lipidic in nature, based on the assumption that viral spikeprotein particles would have appeared exclusively on the
concave fracture face of the liposome.
. They quickly froze influenza
virus and viral receptor-containing liposomes and processed them for EM. At pH 7.4 and 37°C, the viruses did
not fuse with the liposomal membrane, but induced protrusions 35-60 nm in diameter predominantly on the convex liposomal fracture face. Invaginations complementary to the protrusions were predominant on the concave face.
In the center of each protrusion, they observed a welldefined particle 9-14 nm in diameter. They regarded the
particle as a local point-contact site between the viral and
liposomal membranes at neutral pH. At pH 5.4 and 37°C,
they observed many IMPs on the concave liposomal fracture face. Because IMPs with the same appearance were
present exclusively on the E-face of intact viruses, they interpreted these IMPs to be representative of viral spike
protein.
). We believe that each small protrusion with
no observable hole contains an IMP, and that the fusion
intermediate must be a lipid-protein complex.
Fig. 12.
Schematic drawings of HA-mediated fusion
mechanisms. (a) The proposed sequence of fusion
events. Upon acidification,
the viral membrane and the
liposomal membrane are
strongly connected by HA
protein. The hydrophobic
parts of HAs that have penetrated into the liposome are
depicted as small solid circles
at I and I (the hydrophilic
parts of HA are not drawn).
At site I, both membranes
are bent to form a microprotrusion of the lipid bilayer. On the fractured interface
(labeled I
), this part appears
as a small protrusion (arrow
labeled Pr) on the convex liposomal face, and as a pit
containing an IMP (arrow labeled Pt) on the concave
face. At the microprotrusion,
an aqueous pore forms
through the two membranes (site II). On the corresponding fractured interface (labeled II
), a larger protrusion with a small pore at its
center (arrow labeled Po) is observed on the convex liposomal face. Through the dilation process, fusion pores expand into distinct fusion sites. A multi-point fusion on a single viral particle forms an intervening membrane (labeled III) after growth of the fusion sites. (b)
A sectioned profile of the microprotrusion. The diameter of the microprotrusion is only 10-20 nm. For details, see Discussion. (c) A
model of the fusion site surrounded by multiple HA spikes. We observed no spike structures closely surrounding the fusion site (shaded
objects). (d) Schematic drawings of a microprotrusion of the lipid bilayer with an IMP at its center. The centrally located HA spikes, anchored to both the apposing membranes as IMPs, may tug out the membranes to form microprotrusions of the lipid bilayer. (Top) The
small black objects denote the hydrophobic parts of HA anchored to the membranes (hydrophilic parts are not drawn); the rods denote
the transmembrane domains of HA, whereas the circles denote parts of HA which have penetrated into the liposome. (Bottom)
Changes in the fractured interface, corresponding to the changes in the microprotrusion shown in the upper panel. See also Discussion.
[View Larger Version of this Image (34K GIF file)]
).
estimated the diameter of the
initial fusion pore to be <4 nm, and this was suggested to
subsequently expand. Sarkar et al. (1989)
studied the redistribution of a water-soluble fluorescent probe, NBDtaurine, from RBC to HA-expressing cells (GP4F), and
also proposed that the initial fusion pore is no larger than
4.5 nm. The measured diameter of the smallest hole in the protrusions which we observed was <4 nm. We also observed a narrow slit <10 nm wide, bridging the concave
faces of the virus and liposome. Our observations are consistent with the results of Spruce et al. and Sarkar et al.,
and we consider that the smallest hole and the narrow slit
are direct evidence for the initial fusion pore.
).
proposed that the time lag before
fusion is due to the aggregation of HA spikes in the contact zone of the virus-target membrane complex, and proposed a model fusion intermediate in which the fusion site
was surrounded by multiple HA spikes. Similar models
have also been proposed by others (Bentz et al., 1990
;
Bentz et al., 1993
; Guy et al., 1992
; Wilschut and Bron,
1993
). Certain structural features seem common to the
schematic drawings made by these authors and the microprotrusion we observed here (Fig. 12 c). First, HA induces
bending of the apposing membranes, creating protuberances roughly as large as HA spikes. Second, because of
this, the site of membrane contact is a local point only a
few nanometers in diameter. Third, fusion may be initiated without the need for inverted micelles (except in the
model proposed by Bentz et al., 1990
).
, Guy et al. (1992)
, and Wilschut and
Bron (1993)
appear to postulate bending or tilting of the
spikes. However we found no evidence of microprotrusions associated with bent or tilted spikes. Also, when we
examined the structure of viruses on mica under fusioncompetent conditions, it was difficult for us to detect any
drastic change in the overall morphology of the spikes.
Based on these observations, we surmise that part of the
HA spike may penetrate into the target membrane while
the body of the spike is standing upright.
-helix and drives the fusion peptides 100 Å toward the
top of the spike, thus allowing the peptides to insert themselves into the target membrane. Bullough et al. (1994)
demonstrated the extended three-stranded coiled-coil in x-ray crystallographs of soluble trimeric fragments prepared
from the acidic form of bromelain-released HA, thus confirming the model. They also reported a second major
change in the bottom region of HA; the lowest part of the
long
-helix became reversed upside down, which was
thought to shorten the bottom region. Furthermore, Yu et
al. (1994)
used spin-labeled synthetic peptides to demonstrate that a certain part of the long helix next to the fusion
peptide may be inserted into the apposed membrane. All
these data seem to suggest that a considerable portion of
the HA spike with the fusion peptides may be inserted
into the target, while the body of the spike is kept upright
(for reviews see Carr and Kim, 1994
; Hughson, 1995
).
Received for publication 27 February 1997 and in revised form 7 March 1997.
Address all correspondence to Toku Kanaseki, The Department of Cell Biology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu City, Tokyo 183, Japan. Tel.: 81-423-25-3881. Fax: 81-423-21-8678.We gratefully acknowledge the technical assistance of Seiko Muto. We thank Hiroshi Miyamoto for useful discussions.
ESR, elecron spin resonance; HA, hemagglutinin; IMP, intramembrane particle; NA, neuroaminidase.