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Title: Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate}
Article:
Abstract:
The mechanism of membrane fusion by “class II” viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein( E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The ∼55-aa-residue, membrane proximal “stem” can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4°C rather than 37°C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.
Introduction:
Membrane fusion is a critical step for infectious entry of enveloped viruses into cells[1]. A viral “fusion protein” facilitates this process, generally in response to molecular cues specific for the cellular compartment in which viral penetration occurs. For example, dengue and other flaviviruses penetrate from endosomes, following uptake by clathrin-mediated endocytosis[2],[3], and proton binding is the immediate fusion trigger[4]. The flaviviruses are insect-borne agents with positive-strand RNA genomes packaged into compact particles, about 500 Å in diameter[5]. Their fusion protein, known as E, is the principal external protein of the virion. It is made as part of a polyprotein, which includes a “chaperone” protein, designated prM( precursor of M). Cleavage of prM during viral maturation releases most of its ectodomain and promotes formation of a well-ordered lattice of 90 E dimers on the virion surface[6],[7]. When the pH drops below about 6. 2, E undergoes a large-scale conformational rearrangement that includes dissociation of the dimer and reconfiguration of the subunits into trimers( Fig. 1A, B)[8]. At an intermediate stage in this complex molecular reorganization, a hydrophobic “fusion loop” at one end of the extended E subunit inserts into the outer leaflet of the target membrane bilayer[9],[10],[11],[12]. Further rearrangement then draws together the fusion loop and the transmembrane segment anchoring E in the viral membrane. The latter step forces the two membranes together, allowing fusion to ensue. This molecular description of flavivirus fusion derives in part from crystal structures of several flavivirus E proteins in conformations corresponding to prefusion dimers and to postfusion trimers( Fig. 1A)[9],[11],[12],[13],[14]. These structure determinations were made possible by C-terminal truncation of the E ectodomain, through proteolytic treatment or recombinant expression, to increase protein solubility and biochemical stability[15]. These soluble forms of E( “sE”) include only the first 395 of the approximately 445 ectodomain residues; they lack a membrane-proximal region called the “stem”( Fig. 1C) that is substantially conserved among flaviviruses. Intermediate-resolution cryoEM reconstructions show that in the prefusion dimer, the stem segment folds into a helix-loop-helix sandwiched between the rest of the ectodomain and the membrane[7],[16]. In the postfusion conformation, it has been inferred that the stem extends along the edge of domain II, which bears the fusion loop at its tip( Fig. 1)[11]. In the postfusion conformation of E, the fusion loops and the C-terminal transmembrane anchors of all three subunits cluster at one end of the rearranged protein( Fig. 1B). The driving force for pinching together of the two membranes appears to come from contacts made by domain III, as it folds back against domain I at the base of the hairpin, and by the stem with domain II, as it “zips” up along domain II. A consequence of this picture is that interfering with either of these interfaces – for example, by a soluble form of domain III or of the stem – will probably prevent viral fusion. A well-known precedent is T-20/entfuvirtide, a peptide inhibitor of HIV-1 infectivity[17],[18],[19],[20]. T-20 is a soluble fragment of gp41 that blocks the analogous zipping-up step in gp41 refolding. Kielian and co-workers have shown that soluble dengue E-protein domain III can inhibit entry of dengue virus bound at the cell surface when fusion is triggered by transient acidification, and they have made similar observations for the related alphavirus fusion protein, E1[21]. The likely mechanism involves binding of the soluble domain to the extended intermediate structure, thereby aborting the internal fold-back. One report also suggests that a stem peptide can inhibit dengue virus infection in a conventional infectivity assay, but with no mechanistic analysis[22]. The observation is puzzling, because we might expect a site for specific stem-peptide binding to be present only after induction of a conformational transition by the low pH of an endosome and an externally applied peptide to be inactive unless carried into the endosome by some non-specific process. In the work reported here, we show that peptides derived from the dengue E protein stem indeed bind the trimeric, postfusion sE conformer, but not the prefusion dimer. The sequence specificity of this interaction is consistent with our prediction, based on the trimeric sE structure, that such peptides should be able to dock against domain II. We also show that the same stem peptides can inhibit viral infectivity by binding the virion at neutral pH before cell attachment and, presumably, by accompanying the virus particles into endosomes. Hydrophobic interactions between the C-terminal segment of the peptide and the viral membrane contribute to virion binding and hence to inhibiting infection; sequence-specific contacts may have a role in initial binding, in addition to their principal inhibitory contribution when internalized peptide binds rearranging E protein, after the virus has encountered a lowered pH.
Discussion:
We have found that peptides derived from the membrane proximal region of the dengue virus E protein bind specifically to the postfusion conformer of sE, which is a model for a fusion intermediate. These peptides also inhibit viral infectivity – an initially surprising observation because transition of E to its postfusion conformation occurs in the sequestered environment of an endosome. We have shown that the inhibition results from direct interaction of the peptides with virions at neutral pH and hence that the virus carries the peptide with it into the cell. The peptides do not associate with virions at 4°C, suggesting that binding depends on dynamic properties of the viral envelope. We have further shown, using a liposome fusion assay, that the stem-derived peptides inhibit viral fusion at a step prior to hemifusion and fusion-pore formation. How do these stem peptides inhibit fusion if their specific binding sites are not exposed until acidification in an endosome? One possible model is that the peptides bind to transiently exposed sites as the E protein fluctuates among an ensemble of conformations. A similar explanation has been offered to explain neutralization of dengue virus by antibodies for which the epitopes are blocked( or partly blocked) on the virion as modeled from the cryoEM structure[30]. The antibodies can trap a small fluctuation of the protein from its equilibrium conformation. The absence of peptide binding to dengue virions at low temperature is consistent with this type of model. Nonetheless, it seems unlikely that E could spontaneously fluctuate at neutral pH toward a conformation close to that of the postfusion trimer( the excursion of the domains and the rearrangement of the E protein are too extensive), and to consider a fluctuation-binding model, it is probably necessary to assume that the peptide associates with a single domain II, rather than with two that are clustered with each other as in the trimer. A small fluctuation could readily make an edge of domain II accessible to peptide: we know domain II to be hinged with respect to domain I and to adopt somewhat different hinge angles in different crystal forms of the sE dimer, suggesting a potential source of conformational heterogeneity[31]. Residual, uncleaved prM, which is present to varying degrees in most dengue inocula, is also likely to cause perturbations in the packing of E dimers on the virion surface[6],[32]. The association of prM with E tilts the latter outward from the particle surface, and we would expect that a small number of prM-E heterodimers would introduce dislocations and local disorder into the otherwise tightly packed lattice of E dimers covering the virion. The results of our membrane-fusion assays with pyrene-labeled virus particles suggest an alternative, two-step model: step 1 is an initial hydrophobic association of peptide and viral membrane, and step 2 is a specific association with E during the low-pH triggered conformational transition( Fig. 7). Even a modest fluctuation of the closely packed E protein on the viral surface would give an exogenous peptide access to the underlying membrane bilayer, of which about two-thirds is free of intimate protein contacts[7]. A peptide that could associate with the membrane, e. g., as an amphipathic helix, would be carried with the virus particle into an endosome and would be available to “spring into place” upon transient exposure of its site on domain II, thereby blocking the fusion-promoting zipping-up of the E-protein stem. Only peptides with the appropriate sequence would be effective, consistent with the properties of the DV2419–447( scram) peptide, which binds well to virions, but not to E trimer, and has no effect on viral infectivity. The two-step model is also consistent with the sequence dependence of peptide binding as measured by the effect of peptides on the intensity of the pyrene excimer in Fig. 6 and with the requirements for trimer binding in vitro as detected by fluorescence polarization( Fig. 2). The last seven residues of DV2419–447 appear to be important for inhibitory activity. This membrane-proximal segment is the most uniformly hydrophobic sequence in the stem. Residues 441–447 contribute substantially to the neutralizing potency of the peptide( compare peptides in Fig. 2) but not to its affinity for the sE trimer. Moreover, reducing their hydrophobicity by substituting the corresponding residues from WNV greatly reduces inhibition of infectivity without affecting trimer binding( data not shown). Thus, these residues might be particularly important contributors to step 1 of the two-step model outlined above. If so, some specific peptide-protein contacts might occur in concert with peptide-lipid association. That is, residues 441–447, which do not contribute to E-protein binding, could associate with the membrane, while some part of the rest of the peptide made contact with its site on E, exposed by fluctuation or disorder as suggested in the single-step model. According to this combination of the two models above, non-specific membrane interactions and specific domain-II interactions would jointly recruit the peptides, rather than functioning only sequentially. Additional specific interactions could then form upon full exposure of domain II during the low-pH induced conformational transition. Our data suggest strategies, which might otherwise have seemed unlikely, for inhibiting entry of viruses that fuse in endosomes or other internal compartments. Peptides from the membrane-proximal region of the HIV gp41 ectodomain are effective inhibitors of HIV entry, but fusion at the cell surface has made the transient intermediate in envelope refolding seem a more plausible target for HIV than for viruses with envelopes that expose an extended intermediate only after internalization[19],[20]. If peptides or small molecules can be concentrated in a viral membrane by tethering to a hydrophobic “tail”, then it may be possible to enhance the potency of inhibitors identified by a simple binding assay such as the one we have used here.
Enveloped viruses must overcome a succession of cellular barriers before establishing infection . One obstacle is fusion of viral and cellular membranes . Rearrangements of proteins on the viral surface facilitate fusion and subsequent delivery of the viral genome into the cytosol . In this study , we probed the fusion-promoting rearrangement of the dengue-virus envelope ( E ) protein . Peptides derived from the membrane proximal “stem” of E bind to a form of recombinant E that represents a late-stage intermediate in its low-pH triggered conformational change . The binding mimics a key step in the fusion-promoting process . We find that these stem peptides also inhibit viral infectivity , with potency proportional to their affinity for E , and that they do so by specifically blocking fusion . We provide evidence that inhibition is a two-step process: an initial , nonspecific interaction of the peptide with the viral membrane , followed by specific binding to E , as the protein undergoes conformational rearrangement . The initial step explains how the virus can carry the peptide into an endosome—a necessary step , because the binding surface on E becomes available only after exposure to low pH . This work extends the model of flavivirus fusion , and suggests strategies for targeting viruses that penetrate from endosomes .