- Email: [email protected]
Membrane rearrangements in fusion mediated by viral proteins
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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 identified at 82' in the S. enterica sv. Typhimuriumchromosome.SPI-3 is located downstreamof selC, the site of insertionof the PAI-1 (pathogenicityisland 1) and LEEislandsin uropathogenicand enteropathogenicstrains of E. coil, respectively,whichsuggestsa commonmechanism 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 characubiquitous event in by fusing their envelopes with cell terization of some viral fusion •cell physiology, is exmembranes. The mechanisms of merger proteins, particularly influenza ploited by enveloped viruses to of lipid bilayers of two membranes hemagglutinin (HA) (for a reenter 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 pore forms and expands. viral membrane with cellular or of membrane merger. In parendosomal membranes have ticular, it is still unclear what G.B. Melikyan * is in the Dept of Molecular been identified 1. The relative comes first in fusion: the mergBiophysics and Physiology,, Rush Medical College, simplicity of the viral fusion 16,53 ing of membrane lipids or the W. Congress Parkway, Chicago, IL 60612, USA; opening of a proteinaceous fumachinery, compared with that L. V. Chernomordik is in the Laboratory of Cellular sion pore that connects the aqueof intracellular fusion, should and Molecular Biophysics, National Institute of Child Health and Human Development, NIH, ous compartments of two memallow us not only to gain an in10 Center Drive, Bethesda, MD 20892-i 855, USA. branes. Here, we will discuss sight into how membranes fuse "tel: +1 312 942 7011, ~zx: +1 312 942 8711, the hypothetical mechanisms of in disparate cell biological proe-mail: [email protected] viral fusion that have mainly cesses but also to design novel emerged from functional studies antiviral drugs. aimed at arresting and characterizing intermediates of Some enveloped viruses (e.g. Sendal and HIV viruses) fusion preceding pore formation and deducing the strucfuse with the plasma membrane at neutral pH, whereas ture of fusion pores from their properties and analyzing others (e.g. influenza virus and baculovirus) enter the cell via an endocytotic pathway 1. In the latter category, the driving forces for fusion pore enlargement. We will concentrate on HA-mediated fusion, which is the the low pH within the endosomal compartment triggers best-characterized biological fusion reaction. the fusion of a viral envelope with an endosomal membrane, allowing the viral nucleocapsid to gain access Fusion - from triggering to pore enlargement to the cytoplasm. In this review, we will mainly focus 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 on fusion reactions in which a well-defined trigger (low pH within endosomes) initiates a transformation in f u s i o n Although biological membrane fusion is controlled by the fusion protein from its initially non-fusogenic conproteins, lipid bilayers must ultimately rearrange to formation to a fusogenic form. Despite detailed struc-
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geometry of a pore in a lipid bilayer (which has more room for lipid polar heads than tails) is favored by lipids with bulky headgroups ('inverted cone-shape'), such as lysophosphatidylcholine (LPC) (Ref. 7). Lipid mixing between contacting membrane leaflets requires stalk formation and, thus, depends mainly on the lipid composition of the contacting membrane leaflets (reviewed in Ref. 8). In contrast, subsequent formation and expansion of a fusion pore depends on the composition of distal leaflets, such that LPC promotes, and PE or c/s-unsaturated fatty acids inhibit, the hemifusion-to-fusion transition.
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Fig. :1.. Structure of influenza hemagglutinin (HA). (a) Schematic representation of the HA trimer, 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 by a trypsin-like protease to acquire fusogenic activity6°. HA1 subunits are shown in blue, HA2 polypeptides are shown in black, and amino-terminal fusion peptides of HA2 that are sequestered in the trimer interface are shown in red. HA1 and HA2 subunits are responsible for membrane binding 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 the fusion peptide and transmembrane domain and contains only a short stretch from the HA1 subunit. Boxes A-D represent (z-helical segments, whereas black dashed lines and red arrows (fusion peptide) represent the segments of the HA2 subunit that are not part of the TBHA2 fragment. Several 15strands identified at the carboxy-terminal end of TBHA2 have been omitted for clarity. Only one 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 against the target membrane. Another noteworthy feature of the low pH conformation of HA2 is a 180 ° inversion of its viral membrane-proximal part (segments C and D)sS. Low pH conformational changes can also lead to inversion of HA2 orientation and insertion of the fusion peptide into the 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, which presumably represents a lower energy state than the native neutral pH conformation.
provide membrane continuity. Pure lipid bilayers fuse under appropriate conditions 2-4. Fusion in this system can be understood within the framework of the stalkpore hypothesis (Fig. 2) 3, which has been substantiated by both theoretical and experimental studies 3's-9. According to this hypothesis, bilayers first merge their contacting leaflets to form a local connection between membranes, termed the 'stalk', which expands into a bilayer septum referred to as the 'hemifusion diaphragm'. Formation and expansion of a pore in the hemifusion diaphragm completes fusion. From the biological point of view, this scheme is appealing because it ensures a 'tight' fusion, without leakage of aqueous content, by' sequentially disturbing and resealing the contacting leaflets and then the inner leaflets. Both stalk formation and fusion pore formation require a transient rearrangement of lipids in both bilayer leaflets from a flat, lamellar structure into highly curved non-bilayer structures (Fig. 2). Different lipids are known to promote different curvatures of lipid leaflets, which correspond to their effective 'molecular shapes' (reviewed in Refs 8,10). The geometry of a stalk intermediate (which has more room for hydrophobic tails than for polar heads of lipids) is favored by lipids with small polar headgroups in relation to the size of their hydrophobic tails, such as phosphatidylethanolamine (PE) and cis-unsaturated fatty acids, which, by convention, are 'cone-shape' lipids. Conversely, the
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Lipid-sensitive stage(s) in biological fusion
The low pH-triggered fusion reactions mediated by viral proteins appear to share several characteristics. Although acidic conditions are required to induce the conformational transition from resting to active fusion proteins 11,12,the pivotal stages of the actual merging of membranes can proceed at neutral pH (Refs 13-16). For HA, establishment of the low pH conformation is associated with insertion of a hydrophobic amino-terminal fusion peptide into the target and viral membranes, as reviewed in Ref. 17 (Figs 1,2). The insertion of the HA peptide into the target membrane anchors this membrane to the viral envelope (Fig. 1) TM. Protein-mediated membrane merger appears to be a multistep reaction 19,2° that involves conformational changes in fusion proteins and association of several fusion protein trimers into a complex 21-2s. Although fusion induced by some viral proteins has specific requirements for small concentrations of particular lipids [for example, fusion mediated by Semliki Forest virus (SFV) E1 glycoprotein26'2v], other membrane lipids in much higher concentrations (5-10 mol%) have a more universal effect on fusion s. For example, LPC inhibits fusion mediated by baculovirus gp64 (Ref. 15), Sendai virus F (Ref. 28), SFV E1 glycoproteins (J. Wilschut, pers. commun.) and influenza virus HA (Refs 16,29,30). In contrast, PE and cis-unsaturated fatty acids (such as oleic acid) promote fusion mediated by HA, E1 and gp64 fusion proteins 1s'16`2v'31. Lipids modulate fusion at a stage that precedes or coincides with membrane merger and fusion pore opening 16but follows the conformational changes in fusion proteins. If LPC is present when contacting cells expressing HA (Ref. 16) or baculovirus gp64 (Ref. 15) are exposed to low pH, fusion is arrested. Subsequent
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removal of LPC, even hours after reneutralizing the medium, allows the fusion reaction to continue. Studies 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). However, at the LPC-arrested stage, the reaction is insensitive to neuraminidase, indicating that binding between membranes is already mediated by the low-pH-induced insertion of the HA fusion peptide into the target membrane. The LPC-arrested stage is sensitive to both proteinase K and thermolysin, which cleave only the low pH conformations of HA (Ref. 18), indicating that activated fusion proteins are still needed for the fusion reaction when the system is released from the LPC-arrested stage.
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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 membrane 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 result, a hemifusion diaphragm is formed by the inner leaflets of two membranes (Fig. 2). Using lipophilic and aqueous fluorescent probes (see Box 1), the mixing 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 continuity 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 hemifusion 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 referred 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 HAinduced fusion 1632'33'37.
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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 resulting 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 triangles 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 chlorpromazine, 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~.
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range of -0.5-5 nS (3-12nm), where the growth of the pore is impaired for a variable period of time before a subsequent quick dilation 38,4°-42 (Fig. 4c). HA-mediated fusion pores can either close (Fig. 4b), a phenomenon known as flicker 35,4°,41,43, or irreversibly dilate. Both transiently and irreversibly opened pores appear to originate and evolve from the same structure 4° (Fig. 4c): they initially have similar chances to close or dilate to a large size. In other words, the fate of a small fusion 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 behavior of fusion pores formed by different 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 membrane. All these conditions are reported to affect the characteristics of fusion pores40,41,44,4.5. Final enlargement of a fusion pore to a diameter that allows release of the nucleocapsid into the cytosol is vital for viral infection. This process is, however, poorly understood. Although RBCs are able to pass a small aqueous dye to HA-expressing cells, they are reported to retain hemoglobin 46 and rhodamine-tagged dextran 36. In addition, syncytia formation, which requires pore diameter to become comparable to cell sizes, is significantly slower than lipid mixing 47. Final enlargement of the fusion pore appears to be controlled by fusion proteins, as suggested by the ability of point mutations in the fusion peptide of HA to impair syncytia formation 48-s°, although pore enlargement can be facilitated by tensions and bending elasticities of fusing membranes 43'sl and cytoskeletal structures ~s2.
Box 1. Experimental techniques to monitor membrane rearrangements during fusion The majority of models used to study mechanisms of viral fusion, including low pH-triggered fusion of viral particles to cells 19,66,67 or liposomes 19,2T and protein-induced cell-cell 12.48.~°.68and cell-planar lipid bilayer ~° fusion systems, are a far cry from the physiological conditions experienced by the virus invading its host cell. However, these systems are readily amenable to an arsenal of experimental techniques. For example, mixing of lipids and aqueous contents during membrane fusion is readily assayed by lipophilic and aqueous fluorescent probes.
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Lipophilic membrane dyes and aqueous dyes (yellow) transfer from a labeled red blood cell (RBC) or liposome (red and blue) to a hemagglutinin (HA)-expressing cell (gray). In addition, the opening and expansion of fusion pores can be simultaneously followed by electrophysiological measurements 25'4°-42, which are able to detect fusion pores of <2 nm in diameter with millisecond time resolution 2~.38. The simplified equivalent electrical circuit for the fusion pore connecting two cells is shown below. The white dashed line represents the boundary between membrane leaflets. The conductance of the fusion Electrical capacitance of pore can be calculated from expericell m e m b r a n e mentally measured electrical admittance of the two fusing celts 69,7°. HA-expressing cells can be readily fused to other target membranes, such as planar lipid bilayers, allowing simultaneous electrical and fluorescence measurements to be conducted and the lipid composition of the target membrane to be fully controlled 33. Fusion pore conductance
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Fusion pores Fusion pores induced by different viral proteins share several features. Most importantly, they appear to be highly dynamic structures that lack distinct and stationary levels of conductances. This is in striking contrast to ionic channels, which usually exhibit welldefined, relatively stable conductance levels. Electrophysiological measurements of fusion pores between HA-expressing cells and RBCs demonstrate that these pores open quickly to small [a fraction of a nanosiemens (nS)], but variable, conductances 38 (Fig. 4). Once opened, fusion pores can double their conductance within about 5 ms (Ref. 38). Fusion pores formed by different viral proteins have remarkably broad distributions of initial conductances 2s'3s'39 (Fig. 4a,b). After opening rapidly, pores grow more slowly, demonstrating a semistable conductance
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Mechanisms of membrane fusion The structure of a nascent fusion pore is a key to understanding the mechanism of fusion. Different models have proposed that this structure is formed by fusion proteins 35,45,lipids or lipid-protein complexes 1'24'33'37'53"s4. Importantly, formation of an entirely proteinaceous pore implies a cascade of intermediates before pore formation that differs from the hemifusion intermediate leading to the formation of a lipidic pore. The proteinaceous fusion pore is assumed to be a barrel-like structure that spans both bilayers and is formed by several fusion proteins 3s'4~. Lipid mixing and, therefore, complete fusion occur only after dissociation of this barrel. It has been shown that the opening of the fusion pore, as detected by electrical measurements, precedes the onset
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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 appears to form as a result of the concerted action of multiple 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 mixing could be strongly delayed in comparison with the actual merger of lipid leaflets. Although it is possible that formation of a proteinaceous 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 conductances of initial fusion pores 2s,39,4°, and the similarities between disparate fusion reactions driven by different fusion proteins 8'37,s5,56are consistent with the 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 effects on viral protein-mediated fusion is explained by the stalk-pore hypothesis of lipid bilayer fusion
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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 proteins that allows energetically unfavorable intermediates to form, leading to fusion. We have used structural rearrangements of lipid bilayers (Fig. 2) as a framework to describe a possible function of fusion proteins. We postulate that the ectodomain of HA increases the propensity of lipids in contacting leaflets to bend into a stalk, whereas the transmembrane domain induces pore formation within the hemifusion diaphragm. Specifically, after the ectodomains of HA undergo low pHinduced conformational changess+,sg, they insert their fusion peptides into the target and viral membranes and force the contacting membrane leaflets to bend towards each other and form a stalk 3's'6. Lateral expansion of the stalk allows the distal leaflets of the membranes to snap together and form a hemifusion diaphragm. The transmembrane domains, driven by conformational changes of the ectodomain, then insert into the hemifusion diaphragm and induce the opening and growth of a lipid fusion pore. Jackknifing of part of the ectodomain proximal to the viral membrane s* (Figs 1,2) can pull the transmembrane segment through the hemifusion diaphragm, resulting in its destabilization. Several essential questions remain to be answered before we can understand how proteins fuse two membranes into one. The functional dissection of fusion into distinct stages, from triggering to final enlargement of the pore, provides important insights into the role(s) of proteins at each stage of the process. We believe that this 'job description' for the fusion proteins will be instrumental in future under-
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Fig, 3. Hemifusion induced by glycosylphosphatidylinositDl (GPI)-Iinked hemagglutinin (HA). Cells expressing wild-type HA (a,c,e) and GPI-HA (b,d,f)
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 membrane dye R18 or a lipophilic fluorescent probe (FM4-64) incorporated only in the inner leaflets of RBC membranes. The distribution of membrane dye (a,b), a small aqueous dye (c,d) and, in another experiment, the inner leaflet probe (e,f) was examined. Wild-type HA induces mixing of both inner and outer membrane dye, as well as aqueous continuity between fusing cells, whereas GPI-HA fails to mediate aqueous and inner leaflet dye mixing. Scale bar = 50 pm. Adapted with permission from Ref. 33.
standing of the molecular rearrangements of proteins and lipids during biological fusion. Questions for future research • 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 tein or by the d y n a m i c transition b e t w e e n initial and final protein conformations? • What occurs earlier in the fusion process: e s t a b l i s h i n g a c o n 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 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 the m e m b r a n e s ? • What are the contributions of fusion proteins and lipids to the 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 fusion site necessary to m e d i a t e m e m b r a n e merger? • 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 parate biological fusion r e a c t i o n s ? • What are the roles of the distinct topological d o m a i n s of fusion 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 brane fusion?
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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. Conductance 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 irreversibly (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 insightfulcomments.Fig.4a was kindlyprovidedby Dr WolfhardAlmers. References 1 Hernandez, L.D. et al. (1996) Annu. Rev. Cell Dev. Biol. 12, 627-661 2 Wilschut,J. (1991) in Membrane Fusion (Wilschut,J. and Hoekstra, D., eds), pp. 89-126, Marcel Dekker 3 Chernomordik, L.V., Melikyan, G.B. and Chizmadzhev,Y.A. (1987) Biochim. Biophys. Acta 906, 309-352 4 Finkelstein, A., Zimmerberg, J. and Cohen, F.S. (1986) Annu. Rev. Physiol. 48, 163-174 5 Kozlov, M.M. and Markin, V.S. (1983) Biofizika 28,255-261 6 Siegel,D.P. (1993) Biophys. J. 65, 2124-2140 7 Chernomordik, L. et al. (1995) Biophys. ]. 69, 922-929 8 Chernomordik, L., Kozlov, M. and Zimmerberg, J. (1995) J. Membr. Biol. 146, 1-14 9 Lee, J. and Lentz, B.R. (1997) Biochemistry 36, 6251-6259 10 Cullis, P.R., Tilcock, C.P. and Hope, M.J. (1991) in Membrane Fusion (Wilschut,J. and Hoekstra, D., eds), pp. 35-64, Marcel Dekker 11 Doms, R.W., Helenius, A. and White, J. (1985)]. Biol. Chem. 260, 2973-2981 12 Blissard, G.W. and Wenz, J.R. (1992) ]. Virol. 66, 6829-6835 13 van Meet, G., Davoust, J. and Simons, K. (1985) Biochemistry
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58 Bullough,P.A. et al. (1994) Nature 371, 37-43 59 Hughson,F.M. (1995) Curr. Opin. Struct. Biol. 5, 507-513 60 Klenk,H-D. et al. (1975} Virology' 68,426-439 61 White,J.M. (1992) Science 258, 917-924 62 Carr, C.M. and Kim,P.S. (1993) Cell 73, 823-832 63 Weber,T. et al. (1994)J. Biol. Chem. 269, 18353-18358 64 Wharton,S.A.et al. (1995) EMBO J. 14, 240-246 65 Chen,J. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12205-12209
66 White,J., Matlin,K. and Helenius,A. (1981)J. Cell Biol. 89, 674-679
67 Puri,A. et al. {1990)J. Virol. 64, 3824-3832 68 Leikina,E., Onaran, H.O. and Zimmerberg,J. (1992) FEBS Lett. 304, 221-224 69 Zimmerberg,J. et al. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1585-1589
70 Breckenridge,L.J. and Almers,W. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1945-1949
Selfish operons and speciation by gene transfer Jeffrey G. Lawrence ne distinctive feature Bacterial genes providing for single arrangement to allow cotranof bacterial genomes is metabolic functions are found in operons, scription; there is no selection the operon, a cluster of possibly because this organization allows for physical proximity without cotranscribed genes that typiefficient horizontal transfer among cotranscription. This rare event organisms. Transferred genes can confer cally provides for a single metamust be strongly selected so bolic function. Models for the novel metabolic phenotypes on their new that when it occurs it is not lost hosts and allow rapid, effective origins of gene clusters can be by genetic drift. Moreover, such exploitation of new environmental niches. rare events must occur repeatdivided into four classes (see edly to place each gene into every Box 1). The 'natal' model Moreover, the mobility of selfish operons may facilitate bacterial speciation. asserts that some gene clusters operon. Genomic rearrangements can perform such feats3; arose by duplication and diJ.G. Lawrence is in the Dept of Biological Sciences, however, three caveats should vergence, and it does not apply of Pinsburgh, Pittsburgh, PA 15260, USA. be noted. First, two genes are to typical bacterial operons. University tel: +1 412 624 4204, fax: +1 412 624 4759, juxtaposed most readily by the Bacterial operons have evolved e-mail: jlawrenc@vms, cis.pitt.edu deletion of the intervening by the assembly of previously DNA: this is not possible when unlinked ancestral genes: the the intervening DNA encodes useful functions. Second, remaining three models describe this process. As disinversions and translocations that bring some genes cussed below, the 'selfish operon' modeP is distinct closer together also serve to disrupt existing gene clusfrom the other models in several ways: (1) it provides a plausible mechanism for the gradual assembly of ters. Third, the necessity for genomic rearrangements can be alleviated if operators providing for co-regulation genes into operons, (2) it provides a selection mechaevolve at unlinked genes I (e.g. the Escherichia coli arg nism both for the assembly of gene clusters and for their maintenance over evolutionary time, (3) it is genes). Both the co-regulation model and the 'Fisher' model consistent with the observation that genes providing also have difficulty explaining the composition of typifor nonessential functions are found in operons and (4) it does not postulate that gene clusters initially cal bacterial operons. The co-regulation model predicts that genes whose co-regulation would be most provided any selective benefit to host organisms. beneficial should be found in operons, but inspection of the E. coli genome reveals that the genes providing Can operons assemble in situ?. for virtually all of the central metabolic processes are Since the discovery of the operon more than 35 years not found in operons. With notable exceptions ~, most ago 2, the regulatory benefit of cotranscription has been operons provide for nonessential functions [e.g. amino assumed to select for operon assembly (see the 'coacid biosynthesis (trp, his, leu) and carbon source utiregulation' model in Box 1). Although co-regulation lization (rnel, lac, pdu)]. Extensions of the Fisher model is an important consequence of operon assembly, and predict that genes encoding coadapted proteins may certainly plays a role in the maintenance of operon orbe clustered to prevent detrimental recombination beganization, it is difficult to explain operon formation tween coadapted alleles4'5. Although this is plausible by selection for cotranscription. For co-regulation to when considering genes whose products physically select for operon assembly, previously unlinked genes interact, it is difficult both to reconcile this model with must be precisely juxtaposed in a single genomic re-
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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 identified at 82' in the S. enterica sv. Typhimuriumchromosome.SPI-3 is located downstreamof selC, the site of insertionof the PAI-1 (pathogenicityisland 1) and LEEislandsin uropathogenicand enteropathogenicstrains of E. coil, respectively,whichsuggestsa commonmechanism 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 characubiquitous event in by fusing their envelopes with cell terization of some viral fusion •cell physiology, is exmembranes. The mechanisms of merger proteins, particularly influenza ploited by enveloped viruses to of lipid bilayers of two membranes hemagglutinin (HA) (for a reenter 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 pore forms and expands. viral membrane with cellular or of membrane merger. In parendosomal membranes have ticular, it is still unclear what G.B. Melikyan * is in the Dept of Molecular been identified 1. The relative comes first in fusion: the mergBiophysics and Physiology,, Rush Medical College, simplicity of the viral fusion 16,53 ing of membrane lipids or the W. Congress Parkway, Chicago, IL 60612, USA; opening of a proteinaceous fumachinery, compared with that L. V. Chernomordik is in the Laboratory of Cellular sion pore that connects the aqueof intracellular fusion, should and Molecular Biophysics, National Institute of Child Health and Human Development, NIH, ous compartments of two memallow us not only to gain an in10 Center Drive, Bethesda, MD 20892-i 855, USA. branes. Here, we will discuss sight into how membranes fuse "tel: +1 312 942 7011, ~zx: +1 312 942 8711, the hypothetical mechanisms of in disparate cell biological proe-mail: [email protected] viral fusion that have mainly cesses but also to design novel emerged from functional studies antiviral drugs. aimed at arresting and characterizing intermediates of Some enveloped viruses (e.g. Sendal and HIV viruses) fusion preceding pore formation and deducing the strucfuse with the plasma membrane at neutral pH, whereas ture of fusion pores from their properties and analyzing others (e.g. influenza virus and baculovirus) enter the cell via an endocytotic pathway 1. In the latter category, the driving forces for fusion pore enlargement. We will concentrate on HA-mediated fusion, which is the the low pH within the endosomal compartment triggers best-characterized biological fusion reaction. the fusion of a viral envelope with an endosomal membrane, allowing the viral nucleocapsid to gain access Fusion - from triggering to pore enlargement to the cytoplasm. In this review, we will mainly focus 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 on fusion reactions in which a well-defined trigger (low pH within endosomes) initiates a transformation in f u s i o n Although biological membrane fusion is controlled by the fusion protein from its initially non-fusogenic conproteins, lipid bilayers must ultimately rearrange to formation to a fusogenic form. Despite detailed struc-
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geometry of a pore in a lipid bilayer (which has more room for lipid polar heads than tails) is favored by lipids with bulky headgroups ('inverted cone-shape'), such as lysophosphatidylcholine (LPC) (Ref. 7). Lipid mixing between contacting membrane leaflets requires stalk formation and, thus, depends mainly on the lipid composition of the contacting membrane leaflets (reviewed in Ref. 8). In contrast, subsequent formation and expansion of a fusion pore depends on the composition of distal leaflets, such that LPC promotes, and PE or c/s-unsaturated fatty acids inhibit, the hemifusion-to-fusion transition.
(c) |
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Fig. :1.. Structure of influenza hemagglutinin (HA). (a) Schematic representation of the HA trimer, 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 by a trypsin-like protease to acquire fusogenic activity6°. HA1 subunits are shown in blue, HA2 polypeptides are shown in black, and amino-terminal fusion peptides of HA2 that are sequestered in the trimer interface are shown in red. HA1 and HA2 subunits are responsible for membrane binding 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 the fusion peptide and transmembrane domain and contains only a short stretch from the HA1 subunit. Boxes A-D represent (z-helical segments, whereas black dashed lines and red arrows (fusion peptide) represent the segments of the HA2 subunit that are not part of the TBHA2 fragment. Several 15strands identified at the carboxy-terminal end of TBHA2 have been omitted for clarity. Only one 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 against the target membrane. Another noteworthy feature of the low pH conformation of HA2 is a 180 ° inversion of its viral membrane-proximal part (segments C and D)sS. Low pH conformational changes can also lead to inversion of HA2 orientation and insertion of the fusion peptide into the 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, which presumably represents a lower energy state than the native neutral pH conformation.
provide membrane continuity. Pure lipid bilayers fuse under appropriate conditions 2-4. Fusion in this system can be understood within the framework of the stalkpore hypothesis (Fig. 2) 3, which has been substantiated by both theoretical and experimental studies 3's-9. According to this hypothesis, bilayers first merge their contacting leaflets to form a local connection between membranes, termed the 'stalk', which expands into a bilayer septum referred to as the 'hemifusion diaphragm'. Formation and expansion of a pore in the hemifusion diaphragm completes fusion. From the biological point of view, this scheme is appealing because it ensures a 'tight' fusion, without leakage of aqueous content, by' sequentially disturbing and resealing the contacting leaflets and then the inner leaflets. Both stalk formation and fusion pore formation require a transient rearrangement of lipids in both bilayer leaflets from a flat, lamellar structure into highly curved non-bilayer structures (Fig. 2). Different lipids are known to promote different curvatures of lipid leaflets, which correspond to their effective 'molecular shapes' (reviewed in Refs 8,10). The geometry of a stalk intermediate (which has more room for hydrophobic tails than for polar heads of lipids) is favored by lipids with small polar headgroups in relation to the size of their hydrophobic tails, such as phosphatidylethanolamine (PE) and cis-unsaturated fatty acids, which, by convention, are 'cone-shape' lipids. Conversely, the
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Lipid-sensitive stage(s) in biological fusion
The low pH-triggered fusion reactions mediated by viral proteins appear to share several characteristics. Although acidic conditions are required to induce the conformational transition from resting to active fusion proteins 11,12,the pivotal stages of the actual merging of membranes can proceed at neutral pH (Refs 13-16). For HA, establishment of the low pH conformation is associated with insertion of a hydrophobic amino-terminal fusion peptide into the target and viral membranes, as reviewed in Ref. 17 (Figs 1,2). The insertion of the HA peptide into the target membrane anchors this membrane to the viral envelope (Fig. 1) TM. Protein-mediated membrane merger appears to be a multistep reaction 19,2° that involves conformational changes in fusion proteins and association of several fusion protein trimers into a complex 21-2s. Although fusion induced by some viral proteins has specific requirements for small concentrations of particular lipids [for example, fusion mediated by Semliki Forest virus (SFV) E1 glycoprotein26'2v], other membrane lipids in much higher concentrations (5-10 mol%) have a more universal effect on fusion s. For example, LPC inhibits fusion mediated by baculovirus gp64 (Ref. 15), Sendai virus F (Ref. 28), SFV E1 glycoproteins (J. Wilschut, pers. commun.) and influenza virus HA (Refs 16,29,30). In contrast, PE and cis-unsaturated fatty acids (such as oleic acid) promote fusion mediated by HA, E1 and gp64 fusion proteins 1s'16`2v'31. Lipids modulate fusion at a stage that precedes or coincides with membrane merger and fusion pore opening 16but follows the conformational changes in fusion proteins. If LPC is present when contacting cells expressing HA (Ref. 16) or baculovirus gp64 (Ref. 15) are exposed to low pH, fusion is arrested. Subsequent
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removal of LPC, even hours after reneutralizing the medium, allows the fusion reaction to continue. Studies 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). However, at the LPC-arrested stage, the reaction is insensitive to neuraminidase, indicating that binding between membranes is already mediated by the low-pH-induced insertion of the HA fusion peptide into the target membrane. The LPC-arrested stage is sensitive to both proteinase K and thermolysin, which cleave only the low pH conformations of HA (Ref. 18), indicating that activated fusion proteins are still needed for the fusion reaction when the system is released from the LPC-arrested stage.
Target membrane I .......: ................:: ..........
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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 membrane 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 result, a hemifusion diaphragm is formed by the inner leaflets of two membranes (Fig. 2). Using lipophilic and aqueous fluorescent probes (see Box 1), the mixing 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 continuity 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 hemifusion 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 referred 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 HAinduced fusion 1632'33'37.
t
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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 resulting 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 triangles 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 chlorpromazine, 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~.
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range of -0.5-5 nS (3-12nm), where the growth of the pore is impaired for a variable period of time before a subsequent quick dilation 38,4°-42 (Fig. 4c). HA-mediated fusion pores can either close (Fig. 4b), a phenomenon known as flicker 35,4°,41,43, or irreversibly dilate. Both transiently and irreversibly opened pores appear to originate and evolve from the same structure 4° (Fig. 4c): they initially have similar chances to close or dilate to a large size. In other words, the fate of a small fusion 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 behavior of fusion pores formed by different 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 membrane. All these conditions are reported to affect the characteristics of fusion pores40,41,44,4.5. Final enlargement of a fusion pore to a diameter that allows release of the nucleocapsid into the cytosol is vital for viral infection. This process is, however, poorly understood. Although RBCs are able to pass a small aqueous dye to HA-expressing cells, they are reported to retain hemoglobin 46 and rhodamine-tagged dextran 36. In addition, syncytia formation, which requires pore diameter to become comparable to cell sizes, is significantly slower than lipid mixing 47. Final enlargement of the fusion pore appears to be controlled by fusion proteins, as suggested by the ability of point mutations in the fusion peptide of HA to impair syncytia formation 48-s°, although pore enlargement can be facilitated by tensions and bending elasticities of fusing membranes 43'sl and cytoskeletal structures ~s2.
Box 1. Experimental techniques to monitor membrane rearrangements during fusion The majority of models used to study mechanisms of viral fusion, including low pH-triggered fusion of viral particles to cells 19,66,67 or liposomes 19,2T and protein-induced cell-cell 12.48.~°.68and cell-planar lipid bilayer ~° fusion systems, are a far cry from the physiological conditions experienced by the virus invading its host cell. However, these systems are readily amenable to an arsenal of experimental techniques. For example, mixing of lipids and aqueous contents during membrane fusion is readily assayed by lipophilic and aqueous fluorescent probes.
Binding
Hemifusion
Fusion
Lipophilic membrane dyes and aqueous dyes (yellow) transfer from a labeled red blood cell (RBC) or liposome (red and blue) to a hemagglutinin (HA)-expressing cell (gray). In addition, the opening and expansion of fusion pores can be simultaneously followed by electrophysiological measurements 25'4°-42, which are able to detect fusion pores of <2 nm in diameter with millisecond time resolution 2~.38. The simplified equivalent electrical circuit for the fusion pore connecting two cells is shown below. The white dashed line represents the boundary between membrane leaflets. The conductance of the fusion Electrical capacitance of pore can be calculated from expericell m e m b r a n e mentally measured electrical admittance of the two fusing celts 69,7°. HA-expressing cells can be readily fused to other target membranes, such as planar lipid bilayers, allowing simultaneous electrical and fluorescence measurements to be conducted and the lipid composition of the target membrane to be fully controlled 33. Fusion pore conductance
T
Fusion pores Fusion pores induced by different viral proteins share several features. Most importantly, they appear to be highly dynamic structures that lack distinct and stationary levels of conductances. This is in striking contrast to ionic channels, which usually exhibit welldefined, relatively stable conductance levels. Electrophysiological measurements of fusion pores between HA-expressing cells and RBCs demonstrate that these pores open quickly to small [a fraction of a nanosiemens (nS)], but variable, conductances 38 (Fig. 4). Once opened, fusion pores can double their conductance within about 5 ms (Ref. 38). Fusion pores formed by different viral proteins have remarkably broad distributions of initial conductances 2s'3s'39 (Fig. 4a,b). After opening rapidly, pores grow more slowly, demonstrating a semistable conductance
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Mechanisms of membrane fusion The structure of a nascent fusion pore is a key to understanding the mechanism of fusion. Different models have proposed that this structure is formed by fusion proteins 35,45,lipids or lipid-protein complexes 1'24'33'37'53"s4. Importantly, formation of an entirely proteinaceous pore implies a cascade of intermediates before pore formation that differs from the hemifusion intermediate leading to the formation of a lipidic pore. The proteinaceous fusion pore is assumed to be a barrel-like structure that spans both bilayers and is formed by several fusion proteins 3s'4~. Lipid mixing and, therefore, complete fusion occur only after dissociation of this barrel. It has been shown that the opening of the fusion pore, as detected by electrical measurements, precedes the onset
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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 appears to form as a result of the concerted action of multiple 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 mixing could be strongly delayed in comparison with the actual merger of lipid leaflets. Although it is possible that formation of a proteinaceous 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 conductances of initial fusion pores 2s,39,4°, and the similarities between disparate fusion reactions driven by different fusion proteins 8'37,s5,56are consistent with the 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 effects on viral protein-mediated fusion is explained by the stalk-pore hypothesis of lipid bilayer fusion
(d) %+ +
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¢
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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 proteins that allows energetically unfavorable intermediates to form, leading to fusion. We have used structural rearrangements of lipid bilayers (Fig. 2) as a framework to describe a possible function of fusion proteins. We postulate that the ectodomain of HA increases the propensity of lipids in contacting leaflets to bend into a stalk, whereas the transmembrane domain induces pore formation within the hemifusion diaphragm. Specifically, after the ectodomains of HA undergo low pHinduced conformational changess+,sg, they insert their fusion peptides into the target and viral membranes and force the contacting membrane leaflets to bend towards each other and form a stalk 3's'6. Lateral expansion of the stalk allows the distal leaflets of the membranes to snap together and form a hemifusion diaphragm. The transmembrane domains, driven by conformational changes of the ectodomain, then insert into the hemifusion diaphragm and induce the opening and growth of a lipid fusion pore. Jackknifing of part of the ectodomain proximal to the viral membrane s* (Figs 1,2) can pull the transmembrane segment through the hemifusion diaphragm, resulting in its destabilization. Several essential questions remain to be answered before we can understand how proteins fuse two membranes into one. The functional dissection of fusion into distinct stages, from triggering to final enlargement of the pore, provides important insights into the role(s) of proteins at each stage of the process. We believe that this 'job description' for the fusion proteins will be instrumental in future under-
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Fig, 3. Hemifusion induced by glycosylphosphatidylinositDl (GPI)-Iinked hemagglutinin (HA). Cells expressing wild-type HA (a,c,e) and GPI-HA (b,d,f)
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 membrane dye R18 or a lipophilic fluorescent probe (FM4-64) incorporated only in the inner leaflets of RBC membranes. The distribution of membrane dye (a,b), a small aqueous dye (c,d) and, in another experiment, the inner leaflet probe (e,f) was examined. Wild-type HA induces mixing of both inner and outer membrane dye, as well as aqueous continuity between fusing cells, whereas GPI-HA fails to mediate aqueous and inner leaflet dye mixing. Scale bar = 50 pm. Adapted with permission from Ref. 33.
standing of the molecular rearrangements of proteins and lipids during biological fusion. Questions for future research • 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 tein or by the d y n a m i c transition b e t w e e n initial and final protein conformations? • What occurs earlier in the fusion process: e s t a b l i s h i n g a c o n 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 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 the m e m b r a n e s ? • What are the contributions of fusion proteins and lipids to the 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 fusion site necessary to m e d i a t e m e m b r a n e merger? • 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 parate biological fusion r e a c t i o n s ? • What are the roles of the distinct topological d o m a i n s of fusion 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 brane fusion?
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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. Conductance 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 irreversibly (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 insightfulcomments.Fig.4a was kindlyprovidedby Dr WolfhardAlmers. References 1 Hernandez, L.D. et al. (1996) Annu. Rev. Cell Dev. Biol. 12, 627-661 2 Wilschut,J. (1991) in Membrane Fusion (Wilschut,J. and Hoekstra, D., eds), pp. 89-126, Marcel Dekker 3 Chernomordik, L.V., Melikyan, G.B. and Chizmadzhev,Y.A. (1987) Biochim. Biophys. Acta 906, 309-352 4 Finkelstein, A., Zimmerberg, J. and Cohen, F.S. (1986) Annu. Rev. Physiol. 48, 163-174 5 Kozlov, M.M. and Markin, V.S. (1983) Biofizika 28,255-261 6 Siegel,D.P. (1993) Biophys. J. 65, 2124-2140 7 Chernomordik, L. et al. (1995) Biophys. ]. 69, 922-929 8 Chernomordik, L., Kozlov, M. and Zimmerberg, J. (1995) J. Membr. Biol. 146, 1-14 9 Lee, J. and Lentz, B.R. (1997) Biochemistry 36, 6251-6259 10 Cullis, P.R., Tilcock, C.P. and Hope, M.J. (1991) in Membrane Fusion (Wilschut,J. and Hoekstra, D., eds), pp. 35-64, Marcel Dekker 11 Doms, R.W., Helenius, A. and White, J. (1985)]. Biol. Chem. 260, 2973-2981 12 Blissard, G.W. and Wenz, J.R. (1992) ]. Virol. 66, 6829-6835 13 van Meet, G., Davoust, J. and Simons, K. (1985) Biochemistry
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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 cotranof bacterial genomes is metabolic functions are found in operons, scription; there is no selection the operon, a cluster of possibly because this organization allows for physical proximity without cotranscribed genes that typiefficient horizontal transfer among cotranscription. This rare event organisms. Transferred genes can confer cally provides for a single metamust be strongly selected so bolic function. Models for the novel metabolic phenotypes on their new that when it occurs it is not lost hosts and allow rapid, effective origins of gene clusters can be by genetic drift. Moreover, such exploitation of new environmental niches. rare events must occur repeatdivided into four classes (see edly to place each gene into every Box 1). The 'natal' model Moreover, the mobility of selfish operons may facilitate bacterial speciation. asserts that some gene clusters operon. Genomic rearrangements can perform such feats3; arose by duplication and diJ.G. Lawrence is in the Dept of Biological Sciences, however, three caveats should vergence, and it does not apply of Pinsburgh, Pittsburgh, PA 15260, USA. be noted. First, two genes are to typical bacterial operons. University tel: +1 412 624 4204, fax: +1 412 624 4759, juxtaposed most readily by the Bacterial operons have evolved e-mail: jlawrenc@vms, cis.pitt.edu deletion of the intervening by the assembly of previously DNA: this is not possible when unlinked ancestral genes: the the intervening DNA encodes useful functions. Second, remaining three models describe this process. As disinversions and translocations that bring some genes cussed below, the 'selfish operon' modeP is distinct closer together also serve to disrupt existing gene clusfrom the other models in several ways: (1) it provides a plausible mechanism for the gradual assembly of ters. Third, the necessity for genomic rearrangements can be alleviated if operators providing for co-regulation genes into operons, (2) it provides a selection mechaevolve at unlinked genes I (e.g. the Escherichia coli arg nism both for the assembly of gene clusters and for their maintenance over evolutionary time, (3) it is genes). Both the co-regulation model and the 'Fisher' model consistent with the observation that genes providing also have difficulty explaining the composition of typifor nonessential functions are found in operons and (4) it does not postulate that gene clusters initially cal bacterial operons. The co-regulation model predicts that genes whose co-regulation would be most provided any selective benefit to host organisms. beneficial should be found in operons, but inspection of the E. coli genome reveals that the genes providing Can operons assemble in situ?. for virtually all of the central metabolic processes are Since the discovery of the operon more than 35 years not found in operons. With notable exceptions ~, most ago 2, the regulatory benefit of cotranscription has been operons provide for nonessential functions [e.g. amino assumed to select for operon assembly (see the 'coacid biosynthesis (trp, his, leu) and carbon source utiregulation' model in Box 1). Although co-regulation lization (rnel, lac, pdu)]. Extensions of the Fisher model is an important consequence of operon assembly, and predict that genes encoding coadapted proteins may certainly plays a role in the maintenance of operon orbe clustered to prevent detrimental recombination beganization, it is difficult to explain operon formation tween coadapted alleles4'5. Although this is plausible by selection for cotranscription. For co-regulation to when considering genes whose products physically select for operon assembly, previously unlinked genes interact, it is difficult both to reconcile this model with must be precisely juxtaposed in a single genomic re-
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