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58 Smith,H.W. and Tucker,J.F. (1976}J. Hyg. 76, 97-108
59 Stojiljkovic,I., B~iumler,A.J. and Heffron,F. (1995)J. Bacteriol.
177, 1357-1366
60 Tsolis,R.M. et al. (1996) Infect. Immun. 64, 4549-4556
61 Groisman,E.A.et al. (1992)Proc. Natl. Acad. Sci. U. S. A. 89,
11939-11943
62 De Groote,M.A. et al. (1996) Science 272, 414-417
63 Johnson,K. et al. (1991) Mol. Microbiol. 5, 401-407
64 Germanier,R. and Furer, E. (1971) Infect. Immun. 4, 663-673
65 Collins,L.V.,Attridge,S. and Hackett,J. (1991) Infect. Irnmun.
59, 1079-1085
66 Nalue,N.A. and Lindberg,A.A.(1990) Infect. Immun. 58,
2493-2501
67 Stone,B.J.and Miller,V.L. (1995) MoI. Microbiol. 17, 701-712
68 Behlau,I. and Miller,S.I. (1993)J. Bacteriol. 175, 4475-4484
69 B~iumler,A.J.,Tsolis,R.M. and Heffron,F. (1996) Proc. Natl.
Acad. Sci. U. S. A. 93,279-283
70 Gulig,P.A. et al. (1993) Mol. MicrobioI. 7, 825-830
71 Sanderson,K.E.,Hessd, A. and Rudd, K.E. (1995) Microbiol.
Rev. 59,241-303
72 Galfin,J.E. and Sansonetti,P.J. (1996) in Escherichiacoliand
Salmonella:Cellular and Molecular Biology (2nd edn)
(Neidhardt, F.C.et al., eds), pp. 2757-2773, ASM Press
73 Jones, B.D.,Ghori,N. and Falkow,S. (1994)J. Exp. Med. 180,
15-23
74 Blanc-Potard,A-B.and Groisman,E.A.EMBO J. (in press)
Note added in proof
A new pathogenicityisland,designatedSPI-3,has recentlybeen identi-
fied at 82' in the S. enterica sv. Typhimuriumchromosome.SPI-3 is
located downstreamof selC, the site of insertionof the PAI-1 (patho-
genicityisland 1) and LEEislandsin uropathogenicand enteropatho-
genicstrains of E. coil, respectively,whichsuggestsa commonmecha-
nism for the acquisitionof these sequences74.

Membrane rearrangements in fusion

mediated by viral proteins
Grigory B. Melikyan and Leonid V. Chernomordik

embrane fusion, a Diverse enveloped viruses enter host cells tural and biochemical charac-

M ubiquitous event in
•cell physiology, is ex-
ploited by enveloped viruses to
by fusing their envelopes with cell
membranes. The mechanisms of merger
of lipid bilayers of two membranes
terization of some viral fusion
proteins, particularly influenza
hemagglutinin (HA) (for a re-
enter their host cells. For many mediated by influenza hemagglutinin and view, see Ref. 1; Fig. 1), we have
viruses, specialized envelope other viral fusion proteins apparently a poor understanding of the
glycoproteins (fusion proteins) involve local lipidic connections that molecular mechanisms of the
responsible for the fusion of the evolve into a bilayer septum in which a transient, highly localized events
viral membrane with cellular or pore forms and expands. of membrane merger. In par-
endosomal membranes have ticular, it is still unclear what
been identified 1. The relative G.B. Melikyan * is in the Dept of Molecular comes first in fusion: the merg-
simplicity of the viral fusion 16,53 Biophysics and Physiology,, Rush Medical College, ing of membrane lipids or the
W. Congress Parkway, Chicago, IL 60612, USA;
machinery, compared with that L. V. Chernomordik is in the Laboratory of Cellular opening of a proteinaceous fu-
of intracellular fusion, should and Molecular Biophysics, National Institute of sion pore that connects the aque-
allow us not only to gain an in- Child Health and Human Development, NIH, ous compartments of two mem-
sight into how membranes fuse 10 Center Drive, Bethesda, MD 20892-i 855, USA. branes. Here, we will discuss
"tel: +1 312 942 7011, ~zx: +1 312 942 8711,
in disparate cell biological pro- e-mail: gmelikia@rpslmc.edu the hypothetical mechanisms of
cesses but also to design novel viral fusion that have mainly
antiviral drugs. emerged from functional studies
Some enveloped viruses (e.g. Sendal and HIV viruses) aimed at arresting and characterizing intermediates of
fuse with the plasma membrane at neutral pH, whereas fusion preceding pore formation and deducing the struc-
others (e.g. influenza virus and baculovirus) enter the ture of fusion pores from their properties and analyzing
cell via an endocytotic pathway 1. In the latter category, the driving forces for fusion pore enlargement. We
the low pH within the endosomal compartment triggers will concentrate on HA-mediated fusion, which is the
the fusion of a viral envelope with an endosomal mem- best-characterized biological fusion reaction.
brane, allowing the viral nucleocapsid to gain access
to the cytoplasm. In this review, we will mainly focus Fusion - from triggering to pore enlargement
on fusion reactions in which a well-defined trigger (low T h e lipid r e a r r a n g e m e n t s u n d e r l y i n g m e m b r a n e
pH within endosomes) initiates a transformation in f u s i o n
the fusion protein from its initially non-fusogenic con- Although biological membrane fusion is controlled by
formation to a fusogenic form. Despite detailed struc- proteins, lipid bilayers must ultimately rearrange to
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842X/97/$17.00 PIE S0966-842X{97}01107-4

TRENDS IN MICROBIC)LO{;Y 349 V{)L. 5 NO. 9 SEPTEMBER 1997

R E V I E W S

geometry of a pore in a lipid bi-

(c) layer (which has more room for
(a) 1 ~ (b) _cJ:
; |
lipid polar heads than tails) is
favored by lipids with bulky head-
groups ('inverted cone-shape'),
such as lysophosphatidylcholine
(LPC) (Ref. 7). Lipid mixing
between contacting membrane
HA2 I .--
g
°
J
,
I

,
J
•
s
"D

;
•
leaflets requires stalk formation
and, thus, depends mainly on the

t ......... ~ i-.--21i
: . . . . . . . . . .i.---21-:
..
C.~: ,
M; 1..
lipid composition of the contact-
ing membrane leaflets (reviewed

Cytoplasmic tail
in Ref. 8). In contrast, subsequent
formation and expansion of a
fusion pore depends on the com-
Fig. :1.. Structure of influenza hemagglutinin (HA). (a) Schematic representation of the HA trimer, position of distal leaflets, such
which is assembled from HA monomers that are synthesized as single polypeptide chains (HAO) of
~550 amino acids. Each HA monomer must be cleaved into two disulfide-linked HAl-HA2 subunits
that LPC promotes, and PE or
by a trypsin-like protease to acquire fusogenic activity6°. HA1 subunits are shown in blue, HA2 c/s-unsaturated fatty acids in-
polypeptides are shown in black, and amino-terminal fusion peptides of HA2 that are sequestered hibit, the hemifusion-to-fusion
in the trimer interface are shown in red. HA1 and HA2 subunits are responsible for membrane bind- transition.
ing and membrane fusion, respectively (reviewed in Refs 1,61). (b) Native and (c) low pH structures
of a soluble domain (TBHA2) of the HA2 subunit prepared from the low pH form of the HA ectodomain
by successive digestion with trypsin and thermolysin (Ref. 58). This water-soluble segment lacks
Lipid-sensitive stage(s) in
the fusion peptide and transmembrane domain and contains only a short stretch from the HA1 sub- biological fusion
unit. Boxes A-D represent (z-helical segments, whereas black dashed lines and red arrows (fusion The low pH-triggered fusion re-
peptide) represent the segments of the HA2 subunit that are not part of the TBHA2 fragment. Sev- actions mediated by viral proteins
eral 15strands identified at the carboxy-terminal end of TBHA2 have been omitted for clarity. Only one
appear to share several charac-
monomer of TBHA2 is shown at either pH. The dramatic change in HA2 conformation that occurs at
low pH apparently yields an extended a-helical coiled-coil stem ~8,62,which positions the fusion peptide teristics. Although acidic con-
against the target membrane. Another noteworthy feature of the low pH conformation of HA2 is a ditions are required to induce the
180 ° inversion of its viral membrane-proximal part (segments C and D)sS. Low pH conformational conformational transition from
changes can also lead to inversion of HA2 orientation and insertion of the fusion peptide into the resting to active fusion pro-
viral membrane 1763,e4(not shown). When expressed in Escherichia coil, a water-soluble proteolytic
fragment of HA2 is trimeric but, at neutral pH, it spontaneously assumes a low pH conformation65,
teins 11,12,the pivotal stages of the
which presumably represents a lower energy state than the native neutral pH conformation. actual merging of membranes
can proceed at neutral pH (Refs
13-16). For HA, establishment
provide membrane continuity. Pure lipid bilayers fuse of the low pH conformation is associated with in-
under appropriate conditions 2-4. Fusion in this system sertion of a hydrophobic amino-terminal fusion pep-
can be understood within the framework of the stalk- tide into the target and viral membranes, as reviewed
pore hypothesis (Fig. 2) 3, which has been substantiated in Ref. 17 (Figs 1,2). The insertion of the HA peptide
by both theoretical and experimental studies 3's-9. Ac- into the target membrane anchors this membrane to
cording to this hypothesis, bilayers first merge their con- the viral envelope (Fig. 1) TM. Protein-mediated mem-
tacting leaflets to form a local connection between mem- brane merger appears to be a multistep reaction 19,2°
branes, termed the 'stalk', which expands into a bilayer that involves conformational changes in fusion pro-
septum referred to as the 'hemifusion diaphragm'. For- teins and association of several fusion protein trimers
mation and expansion of a pore in the hemifusion dia- into a complex 21-2s.
phragm completes fusion. From the biological point of Although fusion induced by some viral proteins has
view, this scheme is appealing because it ensures a 'tight' specific requirements for small concentrations of par-
fusion, without leakage of aqueous content, by' sequen- ticular lipids [for example, fusion mediated by Semliki
tially disturbing and resealing the contacting leaflets Forest virus (SFV) E1 glycoprotein26'2v], other mem-
and then the inner leaflets. brane lipids in much higher concentrations (5-10 mol%)
Both stalk formation and fusion pore formation re- have a more universal effect on fusion s. For example,
quire a transient rearrangement of lipids in both bilayer LPC inhibits fusion mediated by baculovirus gp64
leaflets from a flat, lamellar structure into highly curved (Ref. 15), Sendai virus F (Ref. 28), SFV E1 glycopro-
non-bilayer structures (Fig. 2). Different lipids are teins (J. Wilschut, pers. commun.) and influenza virus
known to promote different curvatures of lipid leaflets, HA (Refs 16,29,30). In contrast, PE and cis-unsaturated
which correspond to their effective 'molecular shapes' fatty acids (such as oleic acid) promote fusion mediated
(reviewed in Refs 8,10). The geometry of a stalk inter- by HA, E1 and gp64 fusion proteins 1s'16`2v'31.
mediate (which has more room for hydrophobic tails Lipids modulate fusion at a stage that precedes or co-
than for polar heads of lipids) is favored by lipids incides with membrane merger and fusion pore open-
with small polar headgroups in relation to the size of ing 16but follows the conformational changes in fusion
their hydrophobic tails, such as phosphatidylethanol- proteins. If LPC is present when contacting cells ex-
amine (PE) and cis-unsaturated fatty acids, which, by pressing HA (Ref. 16) or baculovirus gp64 (Ref. 15)
convention, are 'cone-shape' lipids. Conversely, the are exposed to low pH, fusion is arrested. Subsequent

TRENDS iN MIC'ROBIOLO(iY 350 VOL. 5 NO. 9 SEPTEMBER 1997


removal of LPC, even hours after reneutralizing the
medium, allows the fusion reaction to continue. Stud-
ies with monoclonal antibodies that specifically bind
to the neutral pH conformation of baculovirus gp64
fusion protein ~s demonstrate that gp64 undergoes a
low-pH-dependent conformational change before LPC
arrest.
Neuraminidase treatment of HA-expressing cells that
have been fused with pre-bound red blood cells (RBCs)
inhibits fusion, presumably by cleaving the sialic acid
residues from the sialoglycoprotein receptors for the
HA1 subunit on the surface of the RBCs (Ref. 16). How-
ever, at the LPC-arrested stage, the reaction is insensi-
tive to neuraminidase, indicating that binding between
membranes is already mediated by the low-pH-induced
insertion of the HA fusion peptide into the target mem-
brane. The LPC-arrested stage is sensitive to both pro-
teinase K and thermolysin, which cleave only the low
pH conformations of HA (Ref. 18), indicating that acti-
vated fusion proteins are still needed for the fusion reac-
tion when the system is released from the LPC-arrested
stage.
Hemifusion and stunted fusion
When exposed to low pH, the ectodomain of HA
anchored to an external leaflet of a membrane via
glycosylphosphatidylinositol (GPI-HA) induces mem-
brane hemifusion 32'33. Hemifusion is defined as the
merger between contacting leaflets of two membranes
(outer leaflets for cell-cell fusion), leaving the distal
(inner) leaflets and aqueous contents distinct. As a re-
sult, a hemifusion diaphragm is formed by the inner
leaflets of two membranes (Fig. 2). Using lipophilic
and aqueous fluorescent probes (see Box 1), the mix-
ing of lipids and aqueous contents during membrane
fusion can be readily assayed. Consistent with the
hemifusion phenotype, GH-HA mediates lipid
mixing between the external leaflets of RBCs and
GPI-HA-expressing cells without redistribution of
either small aqueous dye or membrane dye inserted
into the inner leaflet of erythrocytes at low pH (Ref.
33) (Fig. 3). Simultaneous electrophysiological and
fluorescent measurements also confirm the hemifusion
phenotype by demonstrating that the membrane con-
tinuity between GPI-HA-expressing cells and planar
bilayer lipid membranes is established without fusion
pore formation 33.
The similarities between the structures of GPI-HA
and wild-type (wt) HA ectodomains, and the similarities
in their conformational changes at low pH (Ref. 34),
suggest that wt HA might induce fusion via a hemi-
fusion intermediate (however, see Ref. 35). Although
the hemifusion stage has not been directly demonstrated
in HA-mediated fusion, we have recently shown that
triggering fusion between wt HA-expressing cells and
RBCs at lower temperature (e.g. room temperature)
may result in an apparent hemifusion phenotype re-
ferred to as 'stunted fusion'36: represented by lipidic dye
mixing without appreciable aqueous dye redistribution.
If stunted fusion represents a hemifusion state, it might
indicate that hemifusion is a true intermediate of wt HA-
induced fusion 1632'33'37.
TRENDS IN MI(2ROBI()L()GY 351
R E V I E W S
Target membrane
I .......: ................:: .......... t
HAI~
Binding
HA2
I .......• ...............• .........|
Viral membrane i~ w
i Carboxy terminus
Stalk
Hemifusion
I
Fusion pore
1
Complete
fusion
Fig. 2. A hypothetical mechanism of hemagglutinin (HA)-mediated membrane
fusion, combining the stalk-pore hypothesis of lipid membrane fusion 3 with
recent structural 58 and functionaP 6,3z33 data on HA-mediated fusion. Lipid
fusion intermediates are shown as stalk (local hemifusion), hemifusion,
reversible opening of a small fusion pore and enlargement of the pore re-
sulting in complete fusion. Non-bilayer lipids that favor the curvature of a
stalk or the opposite curvature of a fusion pore are shown as mauve trian-
gles with larger hydrophobic regions or larger headgroup (yellow) regions,
respectively. The HA1 subunit is only shown schematically by a dotted line
during binding. Only one HA2 monomer of the HA trimer is presented for
clarity. The main structural domains of HA2 are color-coded and correspond
to segments A-D in Fig. 1. The loop region of HA2 (amino acids 5 5 - 7 6 ) is
depicted as a green line or a green box at neutral and low pH, respectively.
This illustrative model is based on the assumptions that (1) formation of
the extended coiled-coil stem 58,62occurs at early stages of fusion, resulting
in insertion of the fusion peptide (double arrow) into the target and viral (not
shown) membranes, thereby inducing hemifusion of two membranes and
(2) a jackknife turn of the membrane-proximal domain of HA2 (red box)~8
occurs later and is responsible for the hemifusion-to-fusion transition by
pulling the transmembrane domain into the hemifusion diaphragm and
inducing opening and subsequent enlargement of the fusion pore.
Complete fusion can be achieved both for hemifusion
and stunted fusion intermediates by treating cells with
membrane-permeable cationic drugs, such as chlor-
promazine, which partition into inner leaflets of cells.
Consistent with the stalk-pore hypothesis, these drugs
appear to induce fusion pore formation by promoting
the bending of inner leaflets into a fusion pore 3~.
VOL. 5 NO. 9 SEPTEMBER 1997
R E V I E W S

range of -0.5-5 nS (3-12nm), where the

Box 1. Experimental techniques to monitor membrane growth of the pore is impaired for a vari-
rearrangements during fusion able period of time before a subsequent
The majority of models used to study mechanisms of viral fusion, including quick dilation 38,4°-42 (Fig. 4c).
low pH-triggered fusion of viral particles to cells 19,66,67 or liposomes 19,2T and HA-mediated fusion pores can either
protein-induced cell-cell 12.48.~°.68and cell-planar lipid bilayer ~° fusion systems, close (Fig. 4b), a phenomenon known as
are a far cry from the physiological conditions experienced by the virus invad- flicker 35,4°,41,43, or irreversibly dilate. Both
ing its host cell. However, these systems are readily amenable to an arsenal transiently and irreversibly opened pores
of experimental techniques. For example, mixing of lipids and aqueous con- appear to originate and evolve from the
tents during membrane fusion is readily assayed by lipophilic and aqueous same structure 4° (Fig. 4c): they initially have
fluorescent probes.
similar chances to close or dilate to a large
Binding Hemifusion Fusion size. In other words, the fate of a small fu-
sion pore is not predetermined. In contrast
to HA-mediated fusion, baculovirus gp64
and SFV E1 fusion protein-induced pores
open irreversibly and exhibit little, if any,
pore flickering2s,39. However, this and other
quantitative differences in the dynamic be-
havior of fusion pores formed by differ-
ent fusion proteins are hard to interpret
without expressing the proteins in the same
host cell, matching their surface densities
and fusing them to the same target mem-
brane. All these conditions are reported
Lipophilic membrane dyes and aqueous dyes (yellow) transfer from a labeled to affect the characteristics of fusion
red blood cell (RBC) or liposome (red and blue) to a hemagglutinin (HA)-express- pores40,41,44,4.5.
ing cell (gray). Final enlargement of a fusion pore to a
In addition, the opening and expansion of fusion pores can be simulta- diameter that allows release of the nucleo-
neously followed by electrophysiological measurements 25'4°-42, which are able capsid into the cytosol is vital for viral in-
to detect fusion pores of <2 nm in diameter with millisecond time resolu- fection. This process is, however, poorly
tion 2~.38. The simplified equivalent electrical circuit for the fusion pore connect-
ing two cells is shown below. The white dashed line represents the boundary
understood. Although RBCs are able to
between membrane leaflets. pass a small aqueous dye to HA-expressing
The conductance of the fusion cells, they are reported to retain hemoglo-
Electrical capacitance of pore can be calculated from experi- bin 46 and rhodamine-tagged dextran 36. In
cell m e m b r a n e mentally measured electrical ad- addition, syncytia formation, which re-
mittance of the two fusing celts 69,7°. quires pore diameter to become compa-
HA-expressing cells can be readily rable to cell sizes, is significantly slower

T
fused to other target membranes, than lipid mixing 47. Final enlargement of
such as planar lipid bilayers, al- the fusion pore appears to be controlled by
lowing simultaneous electrical and fusion proteins, as suggested by the ability
fluorescence measurements to be
conducted and the lipid composi-
of point mutations in the fusion peptide
tion of the target membrane to be of HA to impair syncytia formation 48-s°,
Fusion pore conductance fully controlled 33. although pore enlargement can be facili-
tated by tensions and bending elasticities
of fusing membranes 43'sl and cytoskeletal
Fusion pores structures ~s2.
Fusion pores induced by different viral proteins share
several features. Most importantly, they appear to Mechanisms of membrane fusion
be highly dynamic structures that lack distinct and The structure of a nascent fusion pore is a key to under-
stationary levels of conductances. This is in striking standing the mechanism of fusion. Different models have
contrast to ionic channels, which usually exhibit well- proposed that this structure is formed by fusion pro-
defined, relatively stable conductance levels. Electro- teins 35,45,lipids or lipid-protein complexes 1'24'33'37'53"s4.
physiological measurements of fusion pores between Importantly, formation of an entirely proteinaceous
HA-expressing cells and RBCs demonstrate that these pore implies a cascade of intermediates before pore for-
pores open quickly to small [a fraction of a nano- mation that differs from the hemifusion intermediate
siemens (nS)], but variable, conductances 38 (Fig. 4). leading to the formation of a lipidic pore. The proteina-
Once opened, fusion pores can double their con- ceous fusion pore is assumed to be a barrel-like struc-
ductance within about 5 ms (Ref. 38). Fusion pores ture that spans both bilayers and is formed by several
formed by different viral proteins have remarkably fusion proteins 3s'4~. Lipid mixing and, therefore, com-
broad distributions of initial conductances 2s'3s'39 plete fusion occur only after dissociation of this barrel.
(Fig. 4a,b). After opening rapidly, pores grow more It has been shown that the opening of the fusion pore, as
slowly, demonstrating a semistable conductance detected by electrical measurements, precedes the onset

TRENDS IN MIC'ROBIOLOGY 352 VOL. 5 NO. 9 SEPTEMBER 1997

 R E V I E W S

of lipid mixing between RBCs and HA-expressing

cells42,4s. This result argues against the hypothesis that
lipid continuity (i.e. hemifusion) is established before
fusion pore formation. However, the fusion pore ap-
pears to form as a result of the concerted action of mul-
tiple fusion proteins assembled in a fusion complex21-2s.
Therefore, the diffusion of membrane probes through
lipidic connections at the fusion site could be restricted
by a 'scaffold' of fusion proteins surrounding its-s(Fig. 2).
In other words, experimentally detectable lipid mix-
ing could be strongly delayed in comparison with the
actual merger of lipid leaflets. (d) %+ +
Although it is possible that formation of a proteina-
ceous pore occurs before lipid continuity is established,
the lipid sensitivity of the stage of fusion preceding the
opening of a fusion pore 16,the wide distribution of con-
ductances of initial fusion pores 2s,39,4°, and the simi- t d + ¢
larities between disparate fusion reactions driven by
different fusion proteins 8'37,s5,56are consistent with the ell,
hypothesis that the small fusion pore is, at least partially,
a lipidic structure. A specific correlation between the (,) ° ;
ability of lipids to bend membrane leaflets and their ¢ ,~¢ o,+
effects on viral protein-mediated fusion is explained
by the stalk-pore hypothesis of lipid bilayer fusion
(Fig. 2) 3,57 .
The energetic penalty of bending lipids into fusion
intermediates can be relatively high s,6. HA-mediated
fusion does not depend on an external energy source
(e.g. ATP hydrolysis) and, thus, has to depend only on
energy liberated by HA2 conformational changes at
low pH. Therefore, it is the interplay between lipids and Fig, 3. Hemifusion induced by glycosylphosphatidylinositDl (GPI)-Iinked
proteins that allows energetically unfavorable inter- hemagglutinin (HA). Cells expressing wild-type HA (a,c,e) and GPI-HA (b,d,f)
mediates to form, leading to fusion. We have used struc- were triggered to fuse to red blood cells (RBCs) by brief application of a low
pH solution. RBCs were colabeled with small aqueous dye and either mem-
tural rearrangements of lipid bilayers (Fig. 2) as a frame- brane dye R18 or a lipophilic fluorescent probe (FM4-64) incorporated only
work to describe a possible function of fusion proteins. in the inner leaflets of RBC membranes. The distribution of membrane dye
We postulate that the ectodomain of HA increases the (a,b), a small aqueous dye (c,d) and, in another experiment, the inner leaf-
propensity of lipids in contacting leaflets to bend into a let probe (e,f) was examined. Wild-type HA induces mixing of both inner
and outer membrane dye, as well as aqueous continuity between fusing
stalk, whereas the transmembrane domain induces pore cells, whereas GPI-HA fails to mediate aqueous and inner leaflet dye mix-
formation within the hemifusion diaphragm. Specifi- ing. Scale bar = 50 pm. Adapted with permission from Ref. 33.
cally, after the ectodomains of HA undergo low pH-
induced conformational changess+,sg, they insert their
fusion peptides into the target and viral membranes standing of the molecular rearrangements of proteins
and force the contacting membrane leaflets to bend and lipids during biological fusion.
towards each other and form a stalk 3's'6. Lateral ex-
pansion of the stalk allows the distal leaflets of the
membranes to snap together and form a hemifusion Questions for future research
diaphragm. The transmembrane domains, driven by • Is fusion m e d i a t e d by a specific c o n f o r m a t i o n of the fusion p r o
conformational changes of the ectodomain, then insert tein or by the d y n a m i c transition b e t w e e n initial and final protein
into the hemifusion diaphragm and induce the open- conformations?
ing and growth of a lipid fusion pore. Jackknifing of • What occurs earlier in the fusion process: e s t a b l i s h i n g a c o n
part of the ectodomain proximal to the viral mem- t i n u o u s lipidic c o n n e c t i o n b e t w e e n m e m b r a n e s or the o p e n i n g
brane s* (Figs 1,2) can pull the transmembrane segment of a p r o t e i n a c e o u s fusion pore as an a q u e o u s p a t h w a y across
through the hemifusion diaphragm, resulting in its the m e m b r a n e s ?
destabilization. • What are the contributions of fusion proteins and lipids to the
Several essential questions remain to be answered final pore e n l a r g e m e n t ?
• What is the m i n i m u m n u m b e r of activated fusion proteins at the
before we can understand how proteins fuse two fusion site necessary to m e d i a t e m e m b r a n e merger?
membranes into one. The functional dissection of • Is t h e r e a c o m m o n m e m b r a n e r e a r r a n g e m e n t underlying d i s
fusion into distinct stages, from triggering to final parate biological fusion r e a c t i o n s ?
enlargement of the pore, provides important insights • What are the roles of the distinct topological d o m a i n s of fusion
into the role(s) of proteins at each stage of the p r o t e i n s such as e c t o and t r a n s m e m b r a n e d o m a i n s in m e m
process. We believe that this 'job description' for the brane fusion?
fusion proteins will be instrumental in future under-

T,END+,NM, RO,,OLOO* 353 VOW+.5 NO. 9 SEPTEI ,BER1997

REVIEWS
(a) (b)
2 ms 0.4 s
,c, t 4231-4241
4$
Fig. 4. Properties of hemagglutinin (HA)-mediated fusion pores.
(a) An averaged conductance profile of fusion pores formed
between HA-expressing fibroblasts and red blood cells. Con-
ductance is shown in picosiemens (pS). 250 pS corresponds
to a pore diameter of ~2.2 nm and a length of ~15 nm. Adapted
with permission from Ref. 38. (b)Typical pattern of a transient
fusion pore formed between an HA-expressing fibroblast and a
planar lipid bilayer. (c) Transient (thick solid line) and irrevers-
ibly (dotted line) opened pores may have virtually overlapping
pathways at early stages of their evolution (pores are aligned
at the opening)4°. The arrow pointing downwards marks the
point at which the transient and irreversibly opened pores have
diverged. The arrow pointing upwards marks the onset of fast
enlargement of the irreversibly opened pore. Pore conductances
in (b) and (c) have been converted to appropriate diameters.
Adapted, with permission from The Rockefeller University Press,
from Ref. 40.
Acknowledgements
We thank Drs Fredric Cohen and Joshua Zimmerberg for continued
support, numerous stimulating discussions and critical reading of the
manuscript. We also thank Dr David Kingsleyfor critical reading of
the manuscript, and Eugenia Leikina for technical assistance and in-
sightfulcomments.Fig.4a was kindlyprovidedby Dr WolfhardAlmers.
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Selfish operons and speciation

by gene transfer
Jeffrey G. Lawrence

ne distinctive feature Bacterial genes providing for single arrangement to allow cotran-

O of bacterial genomes is
the operon, a cluster of
cotranscribed genes that typi-
metabolic functions are found in operons, scription; there is no selection
possibly because this organization allows
efficient horizontal transfer among
for physical proximity without
cotranscription. This rare event
cally provides for a single meta- organisms. Transferred genes can confer must be strongly selected so
bolic function. Models for the novel metabolic phenotypes on their new that when it occurs it is not lost
origins of gene clusters can be hosts and allow rapid, effective by genetic drift. Moreover, such
divided into four classes (see exploitation of new environmental niches. rare events must occur repeat-
Box 1). The 'natal' model Moreover, the mobility of selfish operons edly to place each gene into every
asserts that some gene clusters may facilitate bacterial speciation. operon. Genomic rearrange-
arose by duplication and di- ments can perform such feats3;
vergence, and it does not apply J.G. Lawrence is in the Dept of Biological Sciences, however, three caveats should
to typical bacterial operons. University of Pinsburgh, Pittsburgh, PA 15260, USA. be noted. First, two genes are
tel: +1 412 624 4204, fax: +1 412 624 4759,
Bacterial operons have evolved e-mail: jlawrenc@vms, cis.pitt.edu juxtaposed most readily by the
by the assembly of previously deletion of the intervening
unlinked ancestral genes: the DNA: this is not possible when
remaining three models describe this process. As dis- the intervening DNA encodes useful functions. Second,
cussed below, the 'selfish operon' modeP is distinct inversions and translocations that bring some genes
from the other models in several ways: (1) it provides closer together also serve to disrupt existing gene clus-
a plausible mechanism for the gradual assembly of ters. Third, the necessity for genomic rearrangements
genes into operons, (2) it provides a selection mecha- can be alleviated if operators providing for co-regulation
nism both for the assembly of gene clusters and for evolve at unlinked genes I (e.g. the Escherichia coli arg
their maintenance over evolutionary time, (3) it is genes).
consistent with the observation that genes providing Both the co-regulation model and the 'Fisher' model
for nonessential functions are found in operons and also have difficulty explaining the composition of typi-
(4) it does not postulate that gene clusters initially cal bacterial operons. The co-regulation model pre-
provided any selective benefit to host organisms. dicts that genes whose co-regulation would be most
beneficial should be found in operons, but inspection
Can operons assemble in situ?. of the E. coli genome reveals that the genes providing
Since the discovery of the operon more than 35 years for virtually all of the central metabolic processes are
ago 2, the regulatory benefit of cotranscription has been not found in operons. With notable exceptions ~, most
assumed to select for operon assembly (see the 'co- operons provide for nonessential functions [e.g. amino
regulation' model in Box 1). Although co-regulation acid biosynthesis (trp, his, leu) and carbon source uti-
is an important consequence of operon assembly, and lization (rnel, lac, pdu)]. Extensions of the Fisher model
certainly plays a role in the maintenance of operon or- predict that genes encoding coadapted proteins may
ganization, it is difficult to explain operon formation be clustered to prevent detrimental recombination be-
by selection for cotranscription. For co-regulation to tween coadapted alleles4'5. Although this is plausible
select for operon assembly, previously unlinked genes when considering genes whose products physically
must be precisely juxtaposed in a single genomic re- interact, it is difficult both to reconcile this model with
Copyright © 1997 Elsevier Science Ltd. All rights reserved. 0966 842X/97/$17.00 PII: S0966-842X(97)01110-4

TRENDS IN MICROBIOLOGY 355 VOL 5 NO. 9 SEPTE2VlBER 1 9 9 7