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Bioscience Reports, Vol. 20, No. 6, 2000 MINI REVIEW Functional Domains within Fusion Proteins: Prospectives for Development of Peptide Inhibitors of Viral Cell Fusion Yechiel Shai1 Receiûed August 1, 2000 The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope and target plasma membrane and is mediated by fusion proteins. Recently, several functional domains within fusion proteins from different viral families were identified. Some are directly involved in conformational changes after receptor binding, as suggested by the recent release of crystallographically determined structures of a highly stable core structure of the fusion proteins in the absence of membranes. However, in the presence of membranes, this core binds strongly to the membrane’s surface and dissociates therein. Other regions, besides the N-terminal fusion peptide, which include the core region and an internal fusion peptide in paramyxoviruses, are directly involved in the actual membrane fusion event, suggesting an ‘‘umbrella’’ like model for the membrane induced conformational change of fusion proteins. Peptides resembling these regions have been shown to have specific antiviral activity, presumably because they interfere with the corresponding domains within the viruses. Overall, these studies shed light into the molecular mechanism of membrane fusion induced by envelope glycoproteins and suggest that fusion proteins from different viral families share common structural and functional motifs. KEY WORDS: Membrane fusion, peptide–lipid interaction, fluorescence, viral fusion inhibitors, antiviral peptides. INTRODUCTION The entry of enveloped viruses into host cells is accomplished by fusion of the viral envelope and target plasma membrane (Hoekstra and Kok, 1989; Stegmann et al., 1989; White, 1990). Membrane fusion is mediated by several glycoproteins present in the viral envelope which help to overcome strong repulsive hydration, steric, and electrostatic barriers. One of them, the fusion protein, is directly involved in the membrane fusion event, i.e., the actual mixing of the viral and cell membranes. Fusion proteins from different viral families (e.g. Paramyxo-, Orthomyxo-, and Retroviridae) share conserved features (White, 1990). Specifically, (i) they are type I integral membrane proteins synthesized as inactive precursors that are cleaved by host-cell proteases to become active; (ii) the newly generated N-terminus contains the fusion peptide, an hydrophobic stretch of amino acids believed to insert and destabilize the membrane, leading to fusion (Durell et al., 1997; Epand, 1998); and 1 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: [email protected] 535 0144-8463兾00兾1200-0535$18.00兾0 2000 Plenum Publishing Corporation 536 Shai (iii) either an heptad repeat adjacent to the N-terminal fusion peptide folds into a protease-resistant trimeric coiled-coil at a certain step during the fusion process (Stegmann et al., 1989; White, 1990; Skehel and Wiley, 1998), or two heptad repeats, one adjacent to the N-terminal fusion peptide and the other to the C-terminal transmembrane domain fold into a trimer of heterodimers such that the interior contains the N-terminal heptad repeat trimer and the outer ring the C-terminal heptad repeats (Fass et al., 1996, Skehel and Wiley, 1998; Baker et al., 1999). However, there are also exceptions to these common features among viral fusion proteins, since no coiled-coils regions have been detected in the E envelope glycoprotein from flavivirus (Rey et al., 1995). ARE THE KNOWN 3D STRUCTURES THE ‘‘FUSION ACTIVE’’ CONFORMATIONS? The recent release of crystallographically determined structures of fragments of the fusion proteins of several viruses in the absence of membrane is helpful for formulating hypotheses concerning mechanisms of membrane fusion (Bullough et al., 1994; Rey et al., 1995; Fass et al., 1996; Chan et al., 1997; Weissenhorn et al., 1997; Malashkevich et al., 1998; Weissenhorn et al., 1998; Baker et al., 1999; Kobe et al., 1999; Malashkevich et al., 1999; Yang et al., 1999). However, these structures might not be the same during the membrane fusion event, since conformational changes of both the viral proteins and the membrane occur. Therefore, the stages leading to the actual fusion event are mostly still unknown. In an effort to shed light on this complex phenomenon, the interaction between model membranes and synthetic peptides that mimic different regions within the envelope proteins of several viruses were studied. The synthetic peptide approach is supported by many studies, some of which showed that non-covalently linked fragments of membrane proteins are biologically active. These studies also revealed that transmembrane segments of membrane proteins can coassemble in a specific manner and that these interactions can be used to predict the functional structure of membrane proteins (reviewed in Lemmon and Engelman, 1994; Von Heijne, 1994; Shai, 1995). The present review will focus on the identification of several functional regions within the fusion proteins of several viruses, which are directly involved in conformational changes and the actual membrane fusion event. Peptides that can interfere with the function of these regions have been shown to be potent inhibitors of viralcell fusion. Their plausible mode of action will also be discussed. THE N-TERMINAL FUSION PEPTIDE DOMAIN The binding of a fusion protein to its receptors induces a conformational change in the glycoprotein that results in exposure of a previously hidden hydrophobic Nterminal domain, named ‘‘fusion peptide’’, and its penetration into the target cell membrane. Fusion peptides from different viruses share high homology (Hsu et al., 1981; Gallaher, 1987). Evidence for the role of the fusion peptide domain in mediating membrane fusion comes from mutagenesis studies in intact envelope proteins of several viruses (Bosch et al., 1989; Freed et al., 1992; Delahunty et al., 1996; Functional Domains within Fusion Proteins 537 Pritsker et al., 1999), as well as from studies with synthetic fusion peptides (Lear and DeGrado, 1987; Slepushkin et al., 1990; Burger et al., 1991; Clague et al., 1991; Martin et al., 1991; Yeagle et al., 1991; Epand et al., 1994; Nieva et al., 1994; Rapaport & Shai, 1994; Gray et al., 1996; Kliger et al., 1997; Ruiz-Arguello et al., 1998; Peisajovich et al., 2000a). It has been shown that the ability of a particular peptide to induce membrane fusion is highly dependent on its sequence, and most mutations, even of a single amino acid, severely reduced or abolished its fusogenic activity (Delahunty et al., 1996; Durell et al., 1997; Pécheur et al., 1999). This is different from other families of membrane binding peptides such as antibacterial peptides, pore forming cytolytic peptides and surfactants, which also exert their activities by direct interaction with lipid constituents of the cell membrane, but whose sequences can be altered significantly without affecting their biological function. This property might be important towards the development of antiviral drugs based on the fusion peptides. The significance of studying synthetic fusion peptides has been demonstrated by several observations: (i) there is a direct correlation between the effects of mutations in the intact protein and the peptide analogues (Freed et al., 1992; Horvath and Lamb, 1992; Rapaport and Shai, 1994; Pereira et al., 1995; Martin et al., 1996; Kliger et al., 1997; Pritsker et al., 1999), (ii) the fusion activity of synthetic peptides, measured in ûitro, is sensitive to factors (such as pH or the addition of inhibitory agents) that affect the infectivity of the virus in ûiûo (Wharton et al., 1988; Pereira et al., 1997), and (iii) synthetic fusion peptides showed anti-viral activity, suggesting that they can accurately model and interact with functional domains of the viral protein (Slepushkin et al., 1993; Kliger et al., 1997; Pritsker et al., 1999) (see more details in the following paragraphs). Although the detailed molecular mechanism of the actual fusion event is still unknown, insertion of the fusion peptides into the cell (Harter et al., 1989; Stegmann et al., 1989; Tsurudome et al., 1992; White, 1992; Pak et al., 1994), viral (Ruigrok et al., 1988; Wharton et al., 1988), or both membranes (Guy et al., 1992; Hughson, 1995; Stegmann et al., 1995) has been postulated to induce local membrane dehydration (Zimmerberg et al., 1991) and to promote negative curvature in the bilayer (Epand, 1998), factors that can help to overcome the energetic barriers associated with the fusion process. Brasseur and co-workers (Brasseur et al., 1990; Brasseur, 1991) predicted that N-terminal fusion peptides can insert into the membrane with an orientation oblique with respect to the water-membrane interface. This has been shown experimentally for the N-terminal fusion peptides of Influenza (Tatulian et al., 1995; Han et al., 1999), SIV (Epand et al., 1994; Martin et al., 1994; Colotto et al., 1996; Bradshaw et al., 2000), HIV-1 (Martin et al., 1996; Kliger et al., 1997; Pritsker et al., 1999), Bovine Leukemia virus (Voneche et al., 1992), and Sendai virus (Rapaport and Shai, 1994; Ghosh and Shai, 1999). Oblique orientation should increase the hydrophobic volume of the bilayer, thus promoting negative curvature of the membrane (Epand et al., 1994), a factor known to facilitate fusion (Cheetham et al., 1994; Chernomordik et al., 1995; Chernomordik et al., 1997; Epand, 1998). In line of this, Richardson et al., (Richardson et al., 1980) have shown specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. 538 Shai Further studies revealed that such peptides are active only at a high concentration and their activity is related to their ability to modify the membrane rather to interfere with the correct organization of the fusion proteins (Kelsey et al., 1990; Yeagle et al., 1991; Yeagle et al., 1992; Epand et al., 1993; Stegmann, 1993). One of these peptides, carbobenzoxy-D-phenylanine-L-phenylalanine-glycine (ZfFG), was studied in detail in phospholipid membranes. It was suggested that it likely inhibits membrane fusion from the surface of the lipid bilayer, but not by forming a tight, stoichiometric complex with the phospholipids (Yeagle et al., 1992; Dentino et al., 1995). In addition, Apolipoprotein A-I and its amphipathic helix peptide analogues inhibit human immunodeficiency virus-induced syncytium formation probably via interaction with the membrane (Owens et al., 1990). Further studies showed that a 22 amino acid-long fusion peptide inhibited HIV-1 envelope glycoprotein-induced syncytium formation at concentrations of 1 µM (Slepushkin et al., 1993). The effect of the peptide is certainly enhanced as its length increases, since it was found that the 33 aa fusion peptide derived from the N-terminus of gp41 inhibited fusion at a concentration of two orders of magnitude lower than that reported for the 22 amino acid peptide. At peptide concentrations of 0.01 µM, the peptide caused 50% inhibition of fusion of HIV-1 envelope glycoprotein-expressing cells with human CD4 expressing cells, as revealed by fluorescence video imaging microscopy (Kliger et al., 1997). A control peptide resembling the N-terminal modified version of the fusion peptide of Sendai virus, was inactive in the inhibition assay (Kliger et al., 1997). Recent studies have shown that fusion peptides specifically self-associate when inserted into the membrane, and that the level of oligomerization is important for their activity. This has been demonstrated with fusion peptides that adopt both an α -helical secondary structure (Rapaport and Shai, 1994; Ghosh and Shai, 1999; Ghosh et al., 2000) or a β -sheet structure (Pritsker et al., 1998; Pritsker et al., 1999; Peisajovich et al., 2000a). This may explain the inhibitory effect of the 33-mer synthetic fusion peptide of HIV-1, since the peptide could assemble with the fusion peptide domain of the intact protein and as a result interfered with its correct organization. Interestingly, an amino acid substitution that decreased the fusogenic activity of fusion peptides in a model system decreased the size of the oligomers in SDS-PAGE (Kliger et al., 1997; Pritsker et al., 1999). Similarly, such mutations also decreased the activity of the intact virus or cells expressing the corresponding mutated fusion proteins (Freed et al., 1992; Pritsker et al., 1999). For example, mutation of the valine at position 2 in the amino terminal fusion peptide domain of HIV-1 gp41 to glutamic acid resulted in an envelope glycoprotein that dominantly interfered with both syncytium formation and infection mediated by the wild-type HIV-1 envelope glycoprotein (Freed et al., 1992). This interference was not abolished by excess of wild-type glycoprotein, suggesting that a higher-order envelope glycoprotein complex is involved in membrane fusion. The trans-dominant mutation also inhibited HIV-1 envelope glycoprotein-mediated cell fusion when expressed in target cells (Elson et al., 1994). In an attempt to understand this phenomenon, Kliger et al. (Kliger et al., 1997) synthesized a 33-residue fusion peptide corresponding to the V2E mutant. They showed that the mutant peptide is as active as the WT fusion peptide in inhibiting HIV-1 envelope-mediated cell-cell fusion. The mutant peptide Functional Domains within Fusion Proteins 539 could also self- and co-assemble with the WT fusion peptide in the membrane. Interestingly, the WT, but not the V2E mutant, induced liposome aggregation, destabilization, and fusion. Moreover, the V2E mutant inhibited vesicle fusion induced by the WT peptide, probably by forming inactive heteroaggregates. These data suggest that specific interactions mediated by N-terminal fusion peptides are required to form higher order oligomers necessary for membrane fusion (Kliger et al., 1997). Attempts to define the oligomeric state of the HIV-1 envelope glycoprotein have yielded conflicting results. Several reports indicated that the envelope glycoprotein of HIV-1 is a tetramer in its membrane-bound state (Pinter et al., 1989; Schawaller et al., 1989; Earl et al., 1990). On the other hand, other reports revealed that it forms trimers (Weiss et al., 1990; Lu et al., 1995; Weissenhorn et al., 1996). In the gp41 non-fusogenic form, the fusion peptide is buried, thus it is not responsible for the oligomerization of the protein. However, once the envelope glycoproteins are recruited to form a fusion complex, the fusion peptides will self-assemble. The size of the aggregates formed by the WT fusion peptide of HIV-1 and its V2E and F11G mutants was determined by using SDS as a membrane mimetic environment (Kliger et al., 1997; Pritsker et al., 1999), as has been done with several other membrane proteins (Lemmon et al., 1992; Simmerman et al., 1996). SDS-PAGE revealed that the dominant form of the three peptides is a dimer, but that only the WT peptide forms higher order oligomers, likely trimers兾tetramers. The inability of the V2E and F11G mutants to form higher order oligomers may be correlated with their loss of activity. Furthermore, the coassembly of the V2E with the WT peptide strengthens the notion that the inhibition of membrane fusion exhibited by the WT and the V2E mutant occurs via their association with the WT sequence of the intact protein. This inhibition may occur because the V2E mutant decreases the portion of higher order oligomers of WT, or because the V2E mutant forms non-functional high-order oligomers with the WT peptide. Since the V2E mutant gp41 elicits a dominant interfering effect even in the presence of excess wild-type glycoprotein (Freed et al., 1992), the second possibility is a more likely mechanism. Interestingly, fusion peptide chirality was found not to be important for the function of fusion peptides (Pritsker et al., 1998). Natural phospholipds are all Lforms and therefore provide the biological membrane interface with stereospecificity. It has been shown that cell surfaces and phospholipid monolayers can stereospecifically recognize each other (Bohm et al., 1993; Hanein et al., 1994). In addition, peptide chirality has been shown to be crucial for peptide-peptide interaction in solution. Examples include the ribonuclease S-peptide兾S-protein complex (Corigliano et al., 1985), HIV-1 protease兾substrate complex (Milton et al., 1992) and the coiled coil formed by the heptad repeats derived form the F1 fusion protein of Sendai virus (Ghosh et al., 1998). Nevertheless, it was found that the WT fusion peptide of HIV-1 gp41 and its enantiomeric (all D-amino acid) analogue bind equally to phospholipid membranes and have equal potencies in inducing membrane fusion. These findings indicate that the stereospecificity of the fusion peptide is not important for peptide-lipid interaction during the fusion process (Pritsker et al., 1998). That peptide chirality is not a prerequisite for peptide-lipid interaction has been shown in other cases as well. Enantiomers of amphipathic α -helical lytic peptides such as the bee venom melittin and the antimicrobial peptides cecropin and 540 Shai magainin, possess lytic activity indistinguishable from that of the parent molecules (Bessalle et al., 1990; Wade et al., 1990; Merrifield et al., 1995). These enantiomers preserved the amphipatic α -helical structure of the wild type peptides, a structure proposed to be prerequisite for their function. Since the biological function was preserved, the enantiomeric peptides should be organized in the membrane similarly to their parent all L-amino acid peptides. Similar results were obtained with all D-amino acid Androctonin, a β -sheet antimicrobial peptide (Hetru et al., 2000). Strikingly, it was demonstrated that the WT fusion peptide of HIV-1 gp41 associates with its enantiomeric wild-type fusion peptide in the membrane (Pritsker et al., 1998). The D-amino acid peptide inhibits HIV-1 envelope-mediated cell–cell fusion but does not inhibit cell–cell fusion mediated by HIV-2 envelope glycoprotein, indicating that although chirality-independent, the interaction between fusion peptides within the membrane depends on their specific sequence. THE CORE REGION OF FUSION PROTEINS In addition to the fusion peptide domain, many fusion proteins also contain one of more heptad repeat regions which are often adjacent to the fusion sequence or to the transmembrane anchor domain (Gallaher et al., 1989; Chambers et al., 1992). Site-directed mutagenic studies of HIV-1 gp41 and Measles virus F protein indicate that heptad repeats found in these fusion proteins are required for membrane-fusion activity (Buckland et al., 1992; Dubay et al., 1992; Chen et al., 1993; Doms et al., 1993; Reitter et al., 1995). Purified antibodies directed against a peptide derived from a heptad repeat of gp41 effectively inhibit synctyia formation, thus confirming the involvement of this segment in the membrane fusion process (Vanini et al., 1993). These heptad repeats are speculated mainly to assist in the correct folding of the proteins. The oligomeric structures of the heptad-repeat regions of fusion proteins of Influenza (Carr and Kim, 1993), MoMuLV (Fass et al., 1996), HIV (Wild et al., 1994a; Bernstein et al., 1995; Lu et al., 1995; Wild et al., 1995; Chan et al., 1997; Weissenhorn et al., 1997), SIV (Blacklow et al., 1995) and simian parainfluenza virus 5 fusion protein (SV5 F) (Baker et al., 1999) have been determined. Interestingly, similar core structures were found in a retrovirus such as HIV1 and paramyxoviruses such as SV5. Chan et al., (Chan et al., 1997) determined the crystal structure of a complex composed of the N-terminal peptide, N36, and the Cterminal heptad repeat, C34. They found that three N36 helices form an interior, parallel coiled-coil trimer, while three C34 helices pack in an oblique, antiparallel manner into highly conserved, hydrophobic grooves on the surface of this trimer. This structure shows striking similarity to the low pH-induced conformation of influenza hemagglutinin. Baker et al., (Baker et al., 1999) have solved the crystal structure of a fragment of SV5 F virus, revealing a 96 Å long coiled coil surrounded by three antiparallel helices. This structure places the fusion and transmembrane anchor of SV5 F in close proximity with a large intervening domain at the opposite end of the coiled coil. Similar interactions were found between the N-terminal and the C-terminal heptad repeats of Sendai virus (Ghosh et al., 1997; Ben-Efraim et al., 1999). It is worth noting that the ectodomain portion of the paramyxovirus transmembrane proteins is much longer than that of the corresponding segment in Functional Domains within Fusion Proteins 541 e.g. HIV-1 (∼400 and ∼170 amino acid residues, respectively). Thus, the interaction demonstrated between the heptad repeats of paramyxoviruses occurs between segments separated by ∼300 amino acid residues, while there are only ∼60 amino acid residues between the heptad repeats of HIV-1. Most interestingly, the core region has been found to be a target for antiviral peptides. Synthetic peptides corresponding to heptad repeats adjacent to transmembrane domains of several viruses display selective antiviral activity for the virus of origin, despite the fact that they are derived from ectodomains with different lengths. This suggests that they can accurately model and interact with functional domains of the viral protein. More specifically, peptides corresponding to the heptad repeats of HIV-1 (Wild et al., 1992; Jiang et al., 1993; Wild et al., 1993), Sendai virus (Rapaport et al., 1995; Ghosh et al., 1998), Respiratory Syncytial virus (Lambert et al., 1996), Human Parainfluenza virus type 3 (Lambert et al., 1996), Measles virus (Lambert et al., 1996), SV5 (Joshi et al., 1998) and Newcastle Disease virus (Young et al., 1997; Young et al., 1999) have been shown to inhibit the fusion induced by the respective virus, presumably by interfering with the conformational change induced by the binding to the host-cell receptors. It has been speculated that they bind to the N-terminal heptad repeats, before the C-terminal heptad repeats pack into the grooves formed by the coiled coil. In this way, folding into the putative fusion-active confirmation is prevented. For example, a synthetic peptide overlapping the C-terminal amphipathic helical segment of gp41 and its tryptophan-rich sequence (Salzwedel et al., 1999) (DP178, amino acids 638–673) was reported to inhibit virus infection at extremely low concentrations (Wild et al., 1994b). Remarkably, DP178 blocks cell fusion and viral entry at a concentration of less than 2 ng兾 ml in ûitro. Moreover, DP178 was reported to be a new, promising drug for treating HIV-1 infected humans (Kilby et al., 1998). Recently, Weiss and colleagues demonstrated that DP178 binds gp41 and inhibits envelope-mediated membrane fusion, but only after gp120 interacts with cellular receptors (Furuta et al., 1998). In another recent study (Kliger and Shai, 2000), it was found that whereas DP178 perturbs the partial α -helix nature of peptides corresponding to the leucine兾isoleucine zipper sequence (N36 or N51), it cannot perturb the coiled-coil conformation, modeled by the complex of N36 or N51 with C34. Therefore, it was suggested that the already formed heterotrimer coiled-coil is not the target of inhibition by DP178. The later results are consistent with a model in which DP178 acquires its inhibitory activity by binding to an earlier intermediate of gp41, in which the N and C peptide regions are not yet associated, thus allowing DP178 to bind to the leucine兾isoleucine zipper sequence and consequently to inhibit transition to the fusion-active conformation (Chan & Kim, 1998; Weissenhorn et al., 1999; Kliger and Shai, 2000). This model is supported by the results of Blumenthal and co-workers, suggesting that the prehairpin intermediate stage appears to be induced rapidly upon interaction of gp120 with CD4 and the coreceptor, but is then relatively stable for several minutes (Jones et al., 1998; Muñoz-Barroso et al., 1998), allowing DP178 to interact with the exposed leucine兾isoleucine zipper sequence. Recently, Kim and co-workers designed a peptide, IQN17, which properly presents a prominent pocket on the surface of the central trimeric coiled coil within gp41 (Eckert et al., 1999). Utilizing IQN17 and mirror-image phage display, they identified cyclic, D-peptide inhibitors of HIV-1 542 Shai infection that share a sequence motif. A 1.5 Å cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Blumenthal and his colleagues (Muñoz-Barroso et al., 1998) developed a new three-color assay to keep track of the cell into which fluorecent lipids and兾or solutes are redistributed. Lipid and solute redistribution occur as a result of opening a lipidpermissive fusion pore and a solute-permissive fusion pore, respectively. They found that DP178 completely inhibited solute redistribution at 50 ng兾ml, but not lipid redistribution. This difference was maintained up to 6 h of coculture of gp120–41 expressing cells with target cells, indicating that DPA178 can ‘‘clamp’’ the fusion complex in the lipid mixing intermediate for very long time periods. On the other hand the heptad repeat derived from the N-terminal domain was less potent, but with no differences between lipid and aqueous dye redistribution at the different inhibitor concentrations. Interestingly, an opposite trend was found with the heptad repeats of SV5 F protein of paramyxovirus (Joshi et al., 1998). The C-terminal heptad repeat was found to be a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of the SV5 F protein, whereas the N-terminal heptad repeat inhibited cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Recent studies done by Shai and his colleagues (Kliger et al., 2000), reviewed in (Dimitrov, 2000)) showed that a shifted C-terminal heptad repeat derived from gp41 of HIV1 (termed C34 (Chan et al., 1997)) is a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of HIV-1, similarly to what has been found in the cases of SV5, thus strengthening the similarities between retro- and parmyxoviruses. In the same study, structural and functional characterization of both C34 and DP178 suggested that DP178 has a second binding site, likely its corresponding region within the intact gp41 within the membrane milieu (Kliger et al., 2000, reviewed in Dimitrov, 2000). It is interesting to note that DP-107, a synthetic peptide corresponding to the N-terminal heptad repeat of HIV-1 is 1000 times less active than DP-178. Similarly to this result, it was found that the N-terminal heptad repeat of Sendai virus is much less active than its C-terminal heptad repeat (Ghosh and Shai, 1999), thus further supporting the notion that retro- and paramyxoviruses share common structural motifs. Interestingly, however, attachment of the N-terminal fusion peptide to the N-terminal heptad repeat of Sendai virus significantly enhanced its inhibitory activity (Ghosh and Shai, 1999). Studies on the mode of inhibition of the C-terminal heptad repeat of Sendai virus, SV-465, revealed that only the wild type peptide was active, but not its derivatives, one with two heptadic leucines substituted with two alanines, and the second an antiomeric (all D-amino acids) version of SV-465 (Ghosh et al., 1998). This was consistent with the finding that only SV-465 could co-assemble with two other biologically active heptad repeats derived from Sendai virus fusion protein, namely, the N-terminal heptad repeat and a leucine zipper-like region located in the middle of Sendai virus F1 protein. The finding that SV0-465 inhibited equally when added to virions before or after their attachment to cells, and that its inhibitory effect was not time dependent, suggests that SV-465 can disturb a step which occurs after the binding of virions to the target cells. Functional Domains within Fusion Proteins 543 A LEUCINE ZIPPER REGION BETWEEN THE N- AND C-TERMINAL HEPTAD REPEATS OF PARAMYXOVIRUSES Paramyxoviruses contain an additional leucine zipper motif located in between the N- and C-heptad repeats within the F1 fusion protein. The leucine zipper region, designated SV-269 (amino acids 269-107), in the ectodomain of the Sendai virus protein is extremely conserved in the family of paramyxoviruses (Ghosh et al., 1997). Furthermore, most of the heptadic leucines兾isoleucines are conserved in other members of paramyxoviruses, suggesting a similar role in the corresponding fusion proteins. To find a role for this region in Sendai virus mediated red blood cell hemolysis, Ghosh et al., (Ghosh et al., 1997) synthesized SV-269, a 39 amino acid long peptide and fluorescently labeled it. A mutant peptide, MuSV-269, with only two amino acids (one heptadic leucine and a glutamic acid) interchanged in their positions was also synthesized and investigated. Functional studies revealed that the wild type leucine zipper motif, SV-269, is a potent inhibitor of Sendai virus mediated cell fusion. It was also found that SV-269 exists in an oligomeric state in solution whereas the mutant peptide, MuSV-269, does not. Interestingly, it was found that SV-269, but not MuSV-269, coassembled in solution with the N- and C-terminus heptad repeats, however, only with their longer versions that showed antiviral activity. That SV-269 can self- and coassemble with other domains of the Sendai virus fusion protein is supported by the finding that the peptide binds specifically to Sendai virions whereas its mutant MuSV269 did not (Ghosh et al., 1997). Lamb and co-workers have studied this central leucine zipper (amino acids 255293) in the F protein of the paramyxovirus SV5 (Dutch et al., 1999). They found that whereas the N- and C-terminal heptad repeats interact forming a thermostable, alpha-helical trimer of heterodimers, the central leucine zipper interacts also with the C-terminal heptad repeat forming an helical complex; however, this complex is not thermostable. Furthermore, the peptide 255-293 was not able to destabilize the complex formed by the N- and C-terminal heptad repeats. The role of the central leuzine zipper between amino acids 268 and 289 in the structure and function of the Newcastle disease virus (NDV) F protein was explored by Morrison and co-workers (Sergel et al., 2000) by introducing single point mutations into the F gene cDNA. The mutations affected either folding of the protein or its fusion activity. Two mutations, L275A and L282A, likely interfered with folding of the molecule since these proteins were not proteolytically cleaved, were minimally expressed at the cell surface, and formed aggregates. They found that the L268A protein was fusion inactive in the presence or absence of HN protein expression. However, very surprisingly, the mutant L289A protein mediated syncytium formation in the absence of HN protein expression although the HN protein enhanced the fusion activity. These results show that a single amino acid change in the F(1) portion of the NDV F protein can alter the stringent requirement for HN protein expression in syncytium formation. Similar results were obtained by Ito et al., (Ito et al., 2000). The authors analyzed chimeras of HN-dependent and HNindependent SV5 F proteins and showed that, in addition to Pro 22 at the F2 Nterminus, Glu1 31 (located in the N-terminal heptad repeat) and Ala 290 (located in the central heptad repeat) are important for the HN-independent fusion activity of 544 Shai the SV5 F protein. This suggests a possible role for these regions in the interaction of the F and HN proteins. MEMBRANE-INDUCED CONFORMATIONAL CHANGES OF THE FUSION PROTEIN DURING THE FUSION PROCESS Several conformational changes characterize the fate of viral fusion proteins. The posttranslational cleavage results in a conformational change manifested by an increase in the exposed hydrophobicity, which is thought to be related with the exposure of the fusion peptide (Skehel et al., 1982). Binding to the host-cell receptor induces a second change in conformation that is believed to include the insertion of the fusion peptide into the target membrane and the packing of the C-terminal heptad repeats against the grooves of the coiled-coil formed by the N-terminal heptad repeats, resulting in the structure observed by X-ray crystallography or NMR (Bullough et al., 1994; Fass et al., 1996; Chan et al., 1997; Weissenhorn et al., 1997; Malashkevich et al., 1998; Weissenhorn et al., 1998; Baker et al., 1999; Kobe et al., 1999; Malashkevich et al., 1999; Yang et al., 1999). Using electron paramagnetic resonance analysis, Shin and co-workers (Yu et al., 1994; Rabenstein and Shin, 1995) showed that cysteine-substituted peptides comprising the loop region and part of the N-terminal heptad repeat of Influenza virus fusion protein (HA) and the Nterminal heptad repeat of HIV-1 fusion protein (gp41) insert reversibly into phospholipid vesicles. Based on these results, the authors suggested that binding of the N-terminal heptad-repeat regions to the membrane could bring the viral and cell membranes closer together and facilitate fusion. Ben-Efraim et al. (Ben-Efraim et al., 1999) analyzed the interaction between peptides corresponding to the N- and Cterminal heptad repeats from Sendia virus fusion protein, in the presence and in the absence of membranes. They showed that in aqueous environment, the peptides coassemble into an α -helical complex, presumably with a structure similar to that of the core complex of the homologous SV5 fusion protein determined by x-ray crystallography (Baker et al., 1999) and other fusion proteins (Bullough et al., 1994; Fass et al., 1996; Chang et al., 1997; Weissenhorn et al., 1997; Malashkevich et al., 1998; Weissenhorn et al., 1998; Kobe et al., 1999; Malashkevich et al., 1999; Young et al., 1999). However, in the presence of phospholipid membranes, this complex binds strongly to the membrane’s surface and dissociates therein. This led Shai and co-workers to suggest an ‘‘umbrella’’ like model for the unfolding of the F1 protein (Figure 1 upper panel). In agreement with these observations, NMR studies showed that the N-terminal repeat of the homologous Newcastle Disease virus protein has an α -helical structure in SDS, consistent with the idea that it binds parallel to the bilayer, as a monomer, with its hydrophobic face buried in the membrane (Young et al., 1999). Furthermore, Shai and co-workers (Kliger et al., 2000b) found that recombinant proteins corresponding to the ectodomain of HIV-1 fusion protein, but lacking the fusion peptide, bind membranes and consequently undergo a major conformational change. As a result, the protease-resistant core becomes susceptible to proteolytic digestion. Accordingly, they showed that synthetic peptides corresponding to the segments that construct this core oligomerize in aqueous solution, Functional Domains within Fusion Proteins 545 Fig. 1. The ‘‘Umbrella’’ model for the actiûation of the fusion protein of Sendai ûirus. Upper panel: Model without the internal fusion peptide. Binding of the viral surface glycoprotein to cell receptors (Fig. 1, step a) results in a conformational change of the fusion protein, which leads to the formation of a trimeric coiled-coil (Fig. 1, step b). This leads to the exposure and insertion of the N-terminal fusion peptide in the target membrane (Fig. 1, step b), following by dissociation of the core region upon binding to membranes (Fig. 1, step c). Lower Panel: A revised model that includes the internal fusion peptide. The conformational changes due to receptor binding expose both the internal and the N-terminal fusion peptides. Either the N-terminal fusion peptide (Fig. 1, step d) or the internal fusion peptide (Fig. 1, step e) insert into the target membrane first, followed by binding of both the N-terminal and the Cterminal heptad repeats to the membrane. This causes the ‘‘umbrella’’-like opening of the coiledcoil Fig. 1, step f). The membrane-induced conformational change consequently causes the cellular and viral membranes to approach each other. Subsequently, both the N-terminal and the internal fusion peptides induce the actual merging of the membranes. The figure shows also potential targets for the peptides inhibitors. but dissociate upon binding to the membrane. This suggests that both the N-terminal and C-terminal heptad-repeats can assist in bringing the viral and cellular membranes closer, facilitating the subsequent merging. The similarity between what was found in HIV-1, a retrovirus, and in Sendai, a paramyxovirus, suggests that these distantly related viruses share common steps in their fusion mechanism. Studies on the mechanism of inhibition of viral infection by a synthetic peptide (DP-178 or T-20 modeled after a membrane-proximal region located downstream of the C-terminal heptad repeat of HIV-1 gp41, prompted Kliger et al., (Kliger et al., 546 Shai 2000a) to postulate a further conformational change for HIV-1 gp41. They showed that DP178 could block two steps in gp41 conformational cascade at different affinities. The low affinity site represents inhibition of host-cell receptor-induced conformational change, before the coiled coil binds to the membrane (Kliger and Shai, 2000). The high affinity site represents inhibition of the newly postulated change in the oligomerization state of gp41: DP-178 interacts with its corresponding segment in the full-length protein, and thus inhibits the recruitment of several gp41-membrane complexes which leads to fusion pore formation (Kliger et al., 2000a). A SECOND FUSION PEPTIDE IN THE FUSION PROTEINS OF PARAMYXOVIRUSES Orthomyxo- and retrovirus fusion proteins are thought to contain only one N-terminal fusion peptide (Gallaher, 1987; White, 1990), whereas paramyxoviruses contain, in addition to the N-terminal fusion peptide, a second one in the interior of the fusion protein (Peisajovich et al., 2000b). More specifically, very recently, Peisajovich et al. (Peisajovich et al., 2000b) searched for regions other than the Nterminal fusion peptide in the Sendai virus F protein, that could participate in the actual merging of the viral and cellular membranes. The rationale was that the extracellular domain of the fusion protein of Sendai virus consists of more than 560 amino acids (Homma and Ohuchi, 1973; Scheid and Choppin, 1977), and therefore it is unlikely that the whole membrane fusion process is accounted for only by a small N-terminal fusion peptide. Strikingly, they found a region corresponding to amino acids 214-226 highly homologous to the fusion peptide of the HIV-1 and RSV envelope glycoproteins. Synthetic peptides corresponding to this region, as well as to the elongated version of the peptide, were able to induce membrane fusion of large unilamellar vesicles to a substantially higher extent than the N-terminal 33amino acid fusion peptide (Peisajovich et al., 2000b). Furthermore, an homologous peptide from Measles virus F1 protein was also synthesized and found to be active, suggesting that this novel finding is a common feature of paramyxoviruses (Samuel O., and Shai, Y., unpublished results). These findings led Shai and co-workers to postulate a revised model for paramyxovirus-induced membrane fusion (Figure 1, lower panels). According to this model, the conformational changes due to receptor binding expose both the internal and the N-terminal fusion peptides, Although we can not rule out the possibility that the N-terminal fusion peptide inserts into the target membrane first (Fig. 1, step d), the existence of a second fusion peptide consecutive to the N-terminal heptad repeat, located on top of the coiled coil in this conformation, opens the possibility that the initial interaction with the target membrane is achieved by means of the internal fusion peptide, which oligomerizes and adopts an α -helical structure upon membrane binding, as suggested in Fig. 1, step e. In this conformation, the N-terminal fusion peptide might be embedded in the viral membrane, in agreement with the models suggested by (Kozlov and Chernomordik, 1998; Bentz, 2000). The affinity of both the N-terminal and the C-terminal heptad repeats to the membrane causes the ‘‘umbrella’’-like opening of the coiledcoil (Ben-Efraim et al., 1999) Fig. 1, step f). This membrane-induced conformational change consequently causes the cellular and viral membranes to approach each Functional Domains within Fusion Proteins 547 other. Subsequently, both the N-terminal and the internal fusion peptides induce the actual merging of the membranes. Obviously, further experiments are needed to determine whether both fusion-active segments destabilize the same membrane. The location of the internal and the N-terminal fusion peptides, both in the same membrane (Fig. 1, step f) is therefore speculative. The importance of the second fusion peptide in Sendai virus-induced membrane fusion was highlighted by the finding that a synthetic peptide corresponding to this fusogenic region, namely SV-201, inhibits the fusion between the virus and red blood cells (Ghosh and Shai, 1998). Ghosh and Shai (Ghosh et al., 1998; Ghosh and Shai, 1998) found that SV-201 specifically inhibited virus-mediated hemolysis only when added to virions prior to their attachment to red blood cells. Sendai virus-mediated hemagglutinin assay in the presence of SV-201 demonstrated that the peptide does not disturb the binding of virions to the target red blood cells. Mutant peptides were either inactive or only slightly active. In an attempt to understand the role of SV201 in membrane fusion and its mechanism of inhibition, SV-201 and its shorter and longer reversion, as well as several mutants were synthesized and investigated for their inhibitory effect, their fusogenic activity, their structure in the membrane bound state, and their ability to oligomerize in solution and when bound to membranes (Ghosh et al., 2000). The results helped to understand SV-201 mechanism of inhibition and strongly support the ‘‘umbrella’’ model of Sendai-virus induced membrane fusion (Peisajovich et al., 2000b). The differences in the inhibitory activity, their ability to bind to Sendai virions, and the oligomerization state of these peptides in aqueous solution and within the membrane was used to distinguish whether SV-201 inhibits the entry of Sendai virus into the host cell by binding to its counterpart in the F1 protein before or after the internal fusion peptide binds to the membrane. The data revealed that both SV-201 and its inactive short version were able to oligomerize in the membrane, whereas only SV-201 oligomerized in aqueous solution (Ghosh and Shai, 1998; Ghosh et al., 2000; Peisajovich et al., 2000b). Furthermore, only SV-201 interacts in a specific manner with active virions in the absence of target cells, whereas this interaction was significantly reduced after incubation of the virus at 60°C, which results in a conformational change for the F protein (Wharton et al., 2000). If inhibition is accomplished by binding to its corresponding region in the membrane, it would be expected that both peptides are inhibitors. On the other hand, if inhibition is achieved by binding to the corresponding region in aqueous solution, it would be expected that only SV-201 will be an inhibitor. Since SV-201 but not its shorter version inhibits viral entry to RBC, Shai and co-workers postulated that SV-201 binds to its counterpart in the F1 protein before the internal fusion peptide binds to the membrane, thus blocking the receptor-induced conformational change, as depicted in Figure 1. It should be noted that although SV–201 oligomerizes under the conditions used in the inhibition experiment, the oligomerization process is reversible (Ghosh and Shai, 1998); therefore part of the peptides are available to interact with their counterparts in the F1 protein. In summary, several functional domains within fusion proteins from different viral families are involved in conformational changes after receptor binding and in the membrane fusion event. These studies suggest an ‘‘umbrella’’ like model for the 548 Shai Fig. 2. The ‘‘Umbrella’’ model for HIV-1-induced membrane fusion: (a) Native state of HIV-1 envelope glycoprotein. (b) Upon binding of gp120 to cellular receptors, a conformational change in gp41 is induced, allowing an extension of the N-terminal fusion peptide toward the cellular membrane. (c) Subsequently, the leucine兾isoleucine zipper and the C-terminal amphipathic sequences are assembled into a core domain which is stable to enzymatic degradation (see inset SDS PAGE). (d) The affinity of both segments to the membrane causes the opening of this three-hairpin structure. The leucine兾isoleucine zipper and the C-terminal amphipathic sequences bind to the cellularbound core is susceptible to enzymatic degradation (see inset SDS PAGE). Further steps, not all of them defined, are needed for complete fusion. The figure shows also potential targets for the peptides inhibitors inhibitors. membrane induced conformational change of fusion proteins. Peptides resembling these regions have been shown to have specific antiviral activity, presumably because they interfere with the corresponding domains within the viruses (Figure 2, dashed boxes). 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