Conformational Modifications of gB from Herpes Simplex Virus Type 1

Article pubs.acs.org/jmc

Conformational Modifications of gB from Herpes Simplex Virus Type 1 Analyzed by Synthetic Peptides Marco Cantisani,†,‡,∥,▽ Annarita Falanga,†,§,▽ Novella Incoronato,⊥ Luigi Russo,⊥ Alfonso De Simone,# Giancarlo Morelli,†,‡,□ Rita Berisio,□ Massimiliano Galdiero,*,‡,⊥ and Stefania Galdiero*,†,‡,§,□ †

Department of Pharmacy, ‡CIRPEB, and §DFM Scarl, University of Naples “Federico II”, Via Mezzocannone 16, 80134, Napoli, Italy Center for Advanced Materials for Health Care IIT@CRIB, Istituto Italiano di Tecnologia, L.g Barsanti e Matteucci 52, 80125, Napoli, Italy ⊥ Department of Experimental Medicine, II University of Naples, Via De Crecchio 7, 80138, Napoli, Italy # Division of Molecular Biosciences, Imperial College London, SW7 2AZ, U.K. □ Istituto di Biostrutture e Bioimmagini − CNR, Via Mezzocannone 16, 80134, Napoli, Italy ∥

S Supporting Information *

ABSTRACT: Entry of enveloped viruses requires fusion of viral and cellular membranes, driven by conformational changes of viral glycoproteins. The crystallized trimeric glycoprotein gB of herpes simplex virus has been described as a postfusion conformation, and several studies prove that like other class III fusion proteins, gB undergoes a pH-dependent switch between the pre- and postfusion conformations. Using several biophysical techniques, we show that peptides corresponding to the long helix of the gB postfusion structure interfere with the membrane fusion event, likely hampering the conformational rearrangements from the pre- to the postfusion structures. Those peptides represent good candidates for further design of peptidomimetic antagonists capable of blocking the fusion process.

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INTRODUCTION Herpes simplex virus (HSV) is an important human pathogen which is responsible for significant morbidity and mortality worldwide. Herpes viruses are a paradigm for viral entry mediated by a multi-component fusion machinery. HSV enters host cells by fusion of the viral envelope with either the plasma membrane1 or an endosomal membrane,2 and the entry pathway is thought to be determined by both virus3,4 and host cell3−7 factors. In particular, HSV-1 enters cells through fusion of the viral envelope with a cellular membrane in a cascade of molecular interactions involving multiple viral glycoproteins and cellular receptors. The envelope glycoproteins gH/gL, gB, and gD are all essential for the entry process,8,9 and expression of this quartet of glycoproteins induces the fusion of cellular membranes in the absence of virus infection.10 Both gH/gL and gB constitute the core fusion machinery and cooperate to induce the initial lipid destabilization that ends in fusion.11 Peptides derived from the gH ectodomain block virus entry,11 while others have the ability to bind and disrupt model membranes.12−18 The recently solved crystal structure of the gH−gL complex19 indicates that gH may be a fusion regulator. Nevertheless, the crystal structure of the gH−gL complex of Epstein−Barr virus (EBV) presents considerable differences in the structural arrangements of domains, suggesting that the gH−gL complex can undergo dynamic rearrangements.8,20 © 2013 American Chemical Society

gB, the most conserved within the herpes virus family, is involved in virus attachment, penetration, and cell-to-cell spread and has proved to function as a membrane fusogen. gB may undergo large conformational changes to bring about fusion, even though clear evidence for its refolding mechanism is still not available. The structures of gB from HSV-121 and EBV22 showed a fold similar to that of the fusion proteins G of vesicular stomatitis virus (VSV)23 and gp64 of baculovirus.24 Several synthetic gB peptides induced the fusion of large unilamellar vesicles and inhibited herpes virus infection.12,25−27 Therefore, gB is a key fusion protein of herpes viruses. gB belongs to class III fusion proteins, which share similar individual domain structures and contain a central threestranded coiled-coil reminiscent of class I proteins. Whereas class I proteins have an N-terminal fusion peptide, class III proteins contain in domain I two fusion loops which resemble the fusion loop of class II proteins, albeit with different sequences. In particular, the canonical class II fusion loop is entirely composed of hydrophobic amino acids whereas gB fusion loops have both hydrophobic and charged residues.21−24,28 The crystal structure of gB21 is a trimer in which multiple contacts between protomers throughout the molecule contribute to its stability. Each protomer of gB can be Received: May 24, 2013 Published: October 12, 2013 8366

dx.doi.org/10.1021/jm400771k | J. Med. Chem. 2013, 56, 8366−8376

Journal of Medicinal Chemistry

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Table 1. Sequence Alignment of Native and Modified Peptides name

protein residues

sequence

gBh gBhN gBhC gBh1 gBh1m gBhs gBh2 gBh2m gBh3 gBh3m gBh4

500−544 500−523 524−544 503−523 503−523 510−523 510−530 510−530 503−530 503−530 500−535

SIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHELTLWNEARK SIEFARLQFTYNHIQRHVNDMLGR VAIAWCELQNHELTLWNEARK FARLQFTYNHIQRHVNDMLGR FARLQFTYNHIQRHVRDMEGR YNHIQRHVNDMLGR YNHIQRHVNDMLGRVAIAWCE YNHIQRHVNDMLGRVKKAWEE FARLQFTYNHIQRHVNDMLGRVAIAWCE FARLQFTYNHIQRHVNDMLGRVKKAWEE SIEFARLQFTYNHIQRHVNDMLGRVAIAWCELQNHE

Figure 1. Cells were exposed to gBh1, gBh1m, gBhs, and gBh at a concentration of 20, 50, 100, and 200 μM: (A) during attachment and entry (cotreatment); (B) after virus penetration (post-treatment), or alternatively, (C) the virus was preincubated with the peptides for 1 h at 37 °C before addition to the cells (virus pretreatment). Results obtained with the gBh1m in all experiments are reported in panel D. For all treatments, nonpenetrated viruses were inactivated by low-pH citrate buffer after the 45 min incubation with cells at 37 °C. The cells were then incubated for 48 h at 37 °C in DMEM supplemented with CMC, and plaque numbers were scored. Experiments were performed in triplicate, and the percentages of inhibition were calculated with respect to no-peptide control experiments. Error bars represent standard deviations.

divided into five distinct domains: I, base; II, middle; III, core; IV crown; V, arm.21 Domain III contains a trimeric coiled coil and a long nonhelical C-terminal arm packing against the coiled coil in an antiparallel fashion. The gB coil arm complex embeds residues 500 to 544 in the coil and 670 to 695 in the arm regions, with an arrangement that brings the C-terminal transmembrane (TM) domain in proximity with the fusion

loops. Connolly and Longnecker28 have hypothesized that gB refolds in a manner similar to class I fusion proteins and that the packing of the arm against the coiled-coil provides a driving force for gB refolding from a prefusion to a postfusion conformation. Thus, also considering its structural resemblance with the G protein postfusion structure of VSV, the gB structure is believed to correspond a postfusion conforma8367



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tion.8,28 Indeed, the removal of the TM domain to get soluble gB may have caused the spontaneous adoption of a postfusion conformation, as previously observed for other fusion proteins.24,29 Differently from VSV, no structural information is so far available for the prefusion state of gB, although gB is known to undergo conformational changes upon variations of pH.30−34 With the aim of interfering with these conformational changes as a strategy to develop efficient inhibitors of viral fusion, we created a set of peptides based on the sequence of the long helical region present in the crystal structure of gB. This class of drugs has had previous therapeutic success in the form of fuzeon, a drug that targets the HIV fusion protein.35 Our results prove that analogues of the N-terminal region of the gB coiled-coil region are efficient inhibitors of HSV-1 fusion. By combining fluorescence and circular dichroism spectroscopies, surface plasmon resonance, and molecular dynamics (MD) simulations, we characterized the mechanism of action of these inhibitors, which are probably able to trap gB in its prefusion state and hamper the refolding process from pre- to postfusion states. These data make gB peptide analogues attractive candidates for further drug development against HSV and may represent a prototype for other class III viruses.

control (untreated) cells and that of cells exposed to the peptides (data not shown). To test whether gB peptides could affect HSV infectivity, Vero cells were treated with HSV-1 in the presence or absence of each peptide under a range of different conditions as described in Experimental Section. Experiments were carried out to identify which step in the entry process was inhibited by gB-derived peptides. These results are shown in Figure 1. All peptides inhibited HSV infection prior to virus penetration into cells. None of the peptides was active in the postexposure treatment, in which cells were infected for 45 min and peptides were then added to the cultures. Virus preincubation was always the most active experiment; in fact approximately 100% inhibition was observed at peptide concentrations of 100 μM. Figure 1 also shows the results of a dose−response coexposure experiment for gBh1m which clearly shows an IC50 of approximately 60 μM. Fluorescence Assays. The long helical sequence gBh contains tryptophan residues almost in the middle of the sequence. We thus used these residues to probe the interaction of the long helix with the shorter peptides. We compared the fluorescence emission spectra of gBh alone in buffer at pH 7.4 (Figure 2) with that obtained after the addition of the other

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RESULTS Design of Peptides. The long helical segment of gB (peptide gBh in the present study) contains the heptad repeat sequence, which is typical of coiled-coil structures. Previous results from our laboratory have shown that a peptide comprising the N-terminus of the long helical sequence (residues 500 to 523, named gBhN) was more active in inhibition compared to the one comprising the C-terminal side (residues 524 to 544, named gBhC).27 Another significant consideration is the length of the helices; in fact, generally, most active antiviral peptide inhibitors comprise the length of three heptads. Therefore, two sequences were designed, gBh1 (from residue 503 to 523) and gBh2 (from residue 510 to 530), both of which comprise three heptad units with the second peptide shifted by one heptad toward the C-terminus. Peptide gBh1 presents a glycine residue at its C-terminal side which seemed like an optimal point in the sequence to end the peptide, as glycines are not usually found in the middle of long helical sequences; the glycine residue is located in the middle of the peptide gBh2. Peptides gBh3 and gBh4 correspond to longer sequences centered around gBh1, while gBhs represents a shorter version (Table 1). To increase the ability of the peptides to adopt a helical structure, some modifications of the native sequences were performed, where charged glutamic acid and arginine residues were introduced into noncore positions (gBh1m, gBh2m, and gBh3m) so that the spacing (i, i+4) favored the formation of an ion pair in the helical conformation (Table 1). The placement of charged residues at the C-terminus also allowed for easy synthesis, purification, characterization, and use of the peptides in in vitro experiments. In fact, the inclusion of the charged residues at peptide termini improves the aqueous solubility and minimizes aggregation problems.36 Virus Entry Assays. Effect of peptides on virus infectivity was analyzed. In particular, all peptides were screened for their ability to inhibit plaque formation. To confirm that they did not exert toxic effect on cells, monolayers were exposed to a range of peptide concentrations (10, 50, 100, 200, and 500 μM) for 24 h, and cell viability was obtained by an LDH assay. No statistical difference was observed between the viability of

Figure 2. Tryptophan fluorescence spectra in buffer of gBh alone and after the addition of gBh1m (A) and gBh2m (B) at different ratios.

peptides. The fluorescence emission of tryptophan residues can be used to follow changes in its microenvironment; in particular, it increases when the amino acid enters a more hydrophobic environment, and together with an increase in quantum yield, the maximal spectral position may be shifted toward shorter wavelengths (blue shift). For peptide gBh1m, 8368



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theoretical and experimental spectra will be different. Figure 3B shows the results of a CD mixing experiment of gBh and gBh1m; a significant difference between the two spectra is evident, strongly suggesting that the two peptides may interact to produce a structural change when mixed together in solution. The results of the CD experiments correlate well with the results of the infectivity inhibition experiments; in fact, we did not observe significant changes in the spectra when using peptides with a lower inhibition activity (data not shown). Surface Plasmon Resonance. To further quantify the binding affinity between the long helical sequence gBh and peptide gBh1m, which was the most active in inhibition experiments, SPR experiments were performed (Figure 4). To improve the signal-to-noise ratio, peptide gBh was biotinylated and then coupled to the streptavidin of the SA sensor chip.

changes in the spectral properties were observed, suggesting that the tryptophan residues of gBh are located in a less polar environment upon interaction (Figure 2); on the contrary, we are unable to observe any change in the spectral properties of the other peptides. An example of the interaction with gBh2 is given in Figure 2. Circular Dichroism. The secondary structures of gB peptides, that were found active in inhibition experiments, were determined by CD spectroscopy in buffer and TFE. While peptide gBh was already able to adopt an α-helical conformation in buffer,27 all the other peptides showed a random coil conformation, but they were able to switch to a helical conformation upon TFE addition (see peptide gBh1m in Figure 3A). CD studies showed that the modified sequences had improved helical propensities over native sequences.

Figure 4. Sensorgrams of the binding between the immobilized gBh and gBh1m using the SA sensor chip.

The SA sensor chip carries a dextran matrix of immobilized streptavidin which is used for capturing the biotinylated ligand. In our method, biotinylated gBh was immobilized on the sensor chip. SPR detection relies on the affinity of the interaction between a molecule and the ligand attached to the sensor surface. gBh1m was loaded onto the gBh sensor chip to measure the binding affinities. The sensorgrams were obtained at pH 7.4. The experiments were also repeated at lower pHs, but we were unable to obtain reproducible data because of aggregation problems just like those in the circular dichroism results. The sensorgrams obtained at pH 7.4 also presented aggregation problems; in fact, they do not properly converge above 700 nM, but this did not prevent the analysis of the data. Numerical integration of the sensorgrams obtained by plotting RUmax values against time at different gBh1m concentrations (200, 500, 700, 800, and 1000 nM) using a simple kinetic 1:1 binding model did not give a good fit as judged by the X2 ∼ 8. This could result from the existence of a more complex interaction between the two peptides or by nonoptimized experimental conditions. The absence of a mass transport effect was assessed by injecting the same gBh1m solution (500 nM) at different flow rates (ranging from 20 to 80 μL/min) over immobilized gBh. No significant variation in the association phase was observed between 20 and 80 μL/min, suggesting that mass transfer was not a limitation for the evaluation of kinetic parameters in this flow-rate range. A flow rate of 20 μL/min was selected for kinetic experiments.

Figure 3. Circular dichroism spectra gBh1m in buffer and at different percentages of TFE (A). Analysis of interactions between gBh and gBh1m by circular dichroism (B): mixing experiments were performed by comparing the spectrum of the two peptides mixed together at the desired concentrations (experimental spectrum) to the sum of the individual spectra of the peptides (theoretical spectrum).

Circular dichroism was also used to verify the possibility of performing biophysical experiments at various pH. We thus obtained CD spectra of gBh1m in different buffers at pH 7.4, 6.5, and 5.5; these spectra clearly indicate that the peptide has a high tendency to aggregate. Circular dichroism was further employed to determine whether any evidence of interaction could be observed between gBh and gBh1m. If two peptides do not interact, no structural change occurs; therefore, the theoretical (the sum of the two spectra of the noninteracting peptides) and experimental (the spectrum of the mix of the two peptides) spectra will be identical. On the contrary, if two peptides do interact, a structural change of the components may result; therefore, 8369



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According to kinetic analysis with BIAeval 3.1 software, experimental sensorgrams were best fitted to the two-state reaction model. In previous SPR studies,37−40 it has been demonstrated that several protein−protein interactions that occur with a multiple contact mode of binding are best fit by this two-state reaction model; moreover, some coiled-coil dimers can undergo a conformational change.39,41 In fact, this model accounts for a conformational change or rearrangement of the complex, with an increasingly stable complex being formed over time. It corresponds to the following equation: A + B ↔ AB ↔ AB*

The kinetic constants and the related thermodynamic dissociation constant, KD, calculated by the BIAeval software, are reported in Table 2. When the whole binding process was evaluated, it appeared that the KD was approximately 300 nM. Table 2. Kinetic and Thermodynamic Constants for gBh1m Binding to Immobilized gBh Peptides association (ka1, ka2) and dissociation (kd1, kd2) rate constants obtained using the two -state model ka1 kd1 KD1a ka2 kd2 KD2a KDappb

(3.82 ± 0.06) (9.33 ± 0.02) 2.4 × 10−6 (2.36 ± 0.05) (3.42 ± 0.08) 0.14 3.0 × 10−7

× 103 × 10−3

Figure 5. (A) RMSD values computed for the simulation with the AMBER force field. As shown in the inset, the molecule is divided in three regions: zone 1 corresponds to part of domain III (residues 536−575), zone 2 to domain II (residues 365−459), and the link connects the two domains (residues 513−535). (B) Population of the α-helical conformation in the region 503−540, according to the DSSP database. (C) Representative structures throughout the trajectories obtained using the AMBER force field after superposition of domain II (residues). The starting conformation observed in the crystal structure of gB is represented in blue. (D) Analysis of the bending angle throughout the MD simulations using the AMBER force field. The bending angle is defined as the angle generated by the centers of mass of zone 1, zone 2, and link regions and displays a value of 169° in the starting X-ray structure of gB. Representative structures of linear and bent states are drawn. (E) Ribbon representation of the starting model used in MD simulations. The region corresponding to the gBh1M peptide portion is drawn in purple.

× 10−3 × 10−4

KD1 = kd1/ka1. bKDapp = [KA1 × (1 + KA2)]−1 with KA1 = KD1−1 and KA2 = KD2−1.

a

The analysis of the data reported in Table 2 clearly indicate that gBh1m initially binds with gBh with a high affinity; this step is followed by a less stringent rearrangement step (as indicated by the kinetic of the second step) which may correspond to the conformational change. Molecular Dynamics Simulation. MD simulations were carried out with a 2-fold aim: (i) to study the structural basis of the different ability of synthetic peptides in inhibiting viral fusion; (ii) to achieve clues on the dynamic properties of gB. To gain a better reliability of results, we carried out parallel MD simulations (100 ns) using both the OPLS and AMBER force fields. Several structural parameters (root-mean-square deviations (RMSD), total number of hydrogen bonds, α-helical content) were used as diagnostics to monitor the structural features of the structures in the trajectory of the unrestrained simulation. The analysis of RMSDs between the starting model and the trajectory structures shows that the system rapidly evolves (within the first 1000 ps) toward states which display large RMSD values, of about 6−10 Å (Figure 5). Large fluctuations of RMSD values suggest some structural variability of the system. To identify the source of this variability, both global and local parameters of the peptide were analyzed. Analyses of both trajectories (using either OPLS or AMBER force fields) showed that the large overall motions are due to bending of the molecule, with its hinge region located on the central helix (Figure 5). To measure bending, the molecule was split in three regions: zone 1 (domain II, residues 365−461), zone 2 (part of domain III, residues 538−572), and the link connecting them. Consistent with a bending motion of the molecule, low RMSD values characterize the two extremities of the simulated

molecule, zone 1 and zone 2, whereas the link region displays higher RMSDs (Figure 5A). Main hinge regions are located at residues 518 and 525, as computed by checking the α-helical content according to the DSSP dictionary (Figure 5B). The resulting bending movement, which preferentially keeps structures of the trajectories in a plane (Figure 5C), is likely due to the restraints imposed by the arm region encompassing residues 123−144. Molecular bending was monitored by defining a bending angle, formed by centers of mass of zone 1, zone 2, and link regions. Evolution of the bending angle, which ranges between 125° and 180° in the AMBER force field (Figure 5D) and between 104° and 180° using the OPLS force field (Figure S1, Supporting Information), shows a high flexibility of the simulated molecule, which continuously changes between a more linear (angle close to 180°) and more bent state (Figure 5).

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DISCUSSION AND CONCLUSION HSV is a widespread human pathogen responsible for significant morbidity and mortality worldwide and is an example of multicomponent fusion machinery involving several viral glycoproteins and cellular receptors. The envelope 8370



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central helix of domain III in the gB prefusion state and hamper proper conformational changes of gB leading to the postfusion structure, in which the three domain III helices must pack together to form a coiled coil (Figure 6). In addition, it was

glycoprotein gB plays a key role in the entry process, and the development of effective fusion inhibitors is fundamental for potential clinical applications and will aid in understanding how HSV executes fusion. Inhibitors that prevent the fusion function of gB may be developed starting from peptide analogues able to interfere with the membrane fusion event, thus preventing conformational changes that occur as gB refolds to its postfusion form by energetically trapping gB in a prefusion state. The available crystallographic structure of the gB ectodomain describes the postfusion structure composed of three protomers, whose bulk coils around the others with a lefthanded twist.21 The essential role of the coiled-coil region in the assembly of gB postfusion state suggests that the central helix may be an interesting target for the design of inhibitors of viral fusion and may serve as a key factor for understanding the HSV membrane fusion mechanism. This study is focused on the long helix spanning residues 500−544 of the gB ectodomain and on a set of peptides of different lengths derived from this sequence to determine the minimal sequence requirement to elicit inhibition of viral infectivity. The analysis of our previously reported antiviral inhibition data27 relative to a peptide corresponding to the entire helix (gBh) and peptides that comprise its N-terminus (gBhN) and its C-terminus (gBhC) demonstrated that, although the three peptides were all able to inhibit HSV-1 infectivity, the peptide corresponding to the N-terminal side of the helix (gBhN) was more active in infectivity inhibition. It was thus speculated that this side of the helix may be exposed in the prefusion state of gB and could thus be targeted by fusion inhibitor peptides. In addition to peptides with native sequences, peptides with motifs stabilizing the helical structure were produced, a strategy which has proven successful for several coiled-coil class I fusion proteins,42−45 including the HIV glycoprotein gp41.45,46 Inhibition experiments clearly show that the peptides corresponding to sequence 503−523 (gBh1 and gBh1m) were the most active, clearly indicating that the N-terminal side is fundamental for inhibition. Moreover, the modification of gBh1 on the C-terminal side with the introduction of the ion pair positively influences activity. gBh1m is the most active in all the experiments and in particular in the virus pretreatment experiment, indicating that it is able to interact with the virus in its prefusogenic structure. Consistent with functional studies, fluorescence data, circular dichroism, and surface plasmon resonance clearly indicate that gBh1m is the most active in establishing an interaction with gBh. The dissociation constant obtained from SPR is KD = 300 nM, indicating a significant interaction with the long helical sequence in gB. Results of functional assays were also interpreted using MD simulations. These studies clearly show that the molecule is a highly flexible assembly of two relatively rigid motional units with a defined secondary structure. This motion is the main contribution to the principal collective motion (Figure 5) and breaks the 44-residue long helix in domain III in two rigid parts (Figure 5). Both peptides gBh1 and gBh1m differ from all the other peptides in that their sequences cover a region of the helix whose structure is mostly invariable whereas the longer gBh3 and gBh4 overlap with highly flexible regions (Figure 5). Therefore, our data suggest that the N-terminal part in domain III helix is fully exposed in the prefusion state and can be reached by inhibiting peptides. In particular, gBh1 and gBh1m, which exhibit the highest affinity to gBh are likely to bind the

Figure 6. Proposed mechanism of inhibition of viral entry by the gBh1m peptide. gBh1m interacts with the accessible region of domain III central helix and hampers coiled-coil formation to form the postfusion state.

shown that peptides spanning the N-terminal end of the coil (residues 500 to 523) inhibit entry better than peptides derived from the C-terminal end of the helix (residues 524 to 544); these data confirm the fact that peptides derived from the Cterminus of gB arms (residues 681 to 695), which stabilize the N-terminal part of the coiled coil, inhibited entry better than those corresponding to the N-terminal end of the arm (residues 670 to 680).25,28 The necessary availability of domain III central helix for inhibition of viral entry due to gBh1 and gBh1m, supported by MD data, provided structural clues on the refolding mechanism of gB from a prefusion to a postfusion state. Indeed, MD analyses showed that trajectory structures continuously evolve from conformations similar to those observed in the crystal structure of gB to a more “bent” state (Figure 5). Superposition of representative “bent” structures to each protomer of the crystallographic structure yields an open trimer (here denoted as open gB), in an umbrella fashion, with the fusion domain projected toward the target membrane. This conformational state resembles the prefusogenic form in class III fusion protein VSV G, with central helix broken down in two helices. Thus, the structural similarity of HSV gB to VSV G strongly suggests that gB may adopt a similar refolding mechanism even though the ectodomain of the latter is smaller and more compact (Figure 7). VSV G, where both prefusion and postfusion structures are available, undergoes a reversible conformational change at low pH,47 involving an extensive structural reorganization. In fact, the main difference between class III fusion proteins and the other two classes is that the prefusion to postfusion conformational change in class I and II is irreversible,48−50 while it seems to be reversible in class III.23,24,51−54 Similarly, gB undergoes conformational changes in response to low pH,55−57 although a prefusogenic state has not been 8371



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Figure 7. (A) Cartoon and surface model of gB obtained using a representative bent structure of MD trajectories (see Experimental Section) superposed on domain III of the crystal structure of gB. The location of Glu acids in the structure is highlighted. The arrow represents the hypothesized umbrella-like closure of gB to reach a postfusion state. (B) Proposed model of gB evolution from a prefusion to a postfusion state. On analogy with VSV, the model proposes (i) opening of domain I to reach the conformation observed in the postfusion state, (ii) umbrella-like closure of gB with the formation of the long domain III central helix, and (iii) zip up of gB arms on the newly formed coiled coil to bring the TM helix toward the host membrane.

characterized. The crystal structures of VSV G protein53 provide a static representation of the pre- and postfusion conformations, but the transition pathway still remains elusive. Albertini et al.47 propose that the transition from the trimeric prefusion to the trimeric postfusion may take place through transient short-lived monomer intermediates, and thus G trimers dissociate at the viral surface during the structural transition. At higher pH, G proteins present on the viral surface may be in equilibrium between the prefusion trimers and flexible monomers. At lower pH, monomers may adopt elongated conformations with the fusion loops at the top of the glycoprotein, favoring the initial interactions with the target membrane and the TM domain on the opposite side. Finally, the monomers reassociate after fusion to form the postfusion conformation with its typical hairpin structure in which the TM domain and the fusion loops are located on the same side. Consistent with our hypothesis of a similar refolding mechanism, gB postfusogenic trimer contains clusters of acidic residues, also observed in the crystal structure of VSV G.23 Of these, Glu502 and Glu535, located at the two opposite sides of the α-helix sequence (500−544) of domain III, are restricted in close space and, at least in the case of Glu535, form hydrogen bonds (Figure S2, Supporting Information). This feature is not commonly observed in proteins and necessarily requires a protonated state of the acidic residues. By calculating pKa values

of these residues in the structure of gB, we observed that Glu508 and Glu535 are characterized by atypically high pKa values (between 6 and 15, Table 2), consistent with the fact that Glu508 and Glu535 are fully buried in hydrophobic clefts. These characteristics of gB suggest that evolution from a pre- to postfusogenic state may be initiated by partial protonation of these Glu residues, which may be driven by a drop of pH in endosomes. Indeed, negatively charged Glu acids would not allow the formation of the stabilizing trimeric coiled-coil structure. On the basis of our “open gB” model, Glu535 is located at the top of the umbrella; its protonation is likely to be the triggering event, followed by protonation of Glu508 to allow the trimer to fold up upon formation of the central coiled coil (umbrella closure in Figure 7). The high flexibility of gB in its monomeric (high pH) state may explain why, contrary to VSV G, a prefusogenic state of gB was never characterized. However, there is an agreement that a more complex mechanism must also involve gH/gL, although the molecular details of the interactions and the reasons for its requirement still are unknown. Indeed, exposure of HSV virions to low pH after endocytosis is required for infection of some but not all cell types and whether one or more glycoproteins must respond to low pH is still under debate. If low pH can indeed trigger an activating conformational change in gB, several possibilities can be envisaged for how inappropriate membrane fusion might be 8372



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For all treatments, nonpenetrated viruses were inactivated by citrate buffer (pH 3.0) after the 45 min incubation at 37 °C. Monolayers were incubated for 48 h at 37 °C in DMEM supplemented with carboxymethylcellulose (CMC), fixed, and stained with X-gal (5bromo-4-chloro-3-indolyl-β-D-galactopyranoside), and plaque numbers were scored. Experiments were performed in triplicate, and the percentage of inhibition was calculated with respect to no-peptide control experiments. Toxicity. Peptide cytotoxicity was measured by a lactate dehydrogenase (LDH) assay (10, 50, 100, 200, 250, and 500 μM) and was carried out according to the manufacturer’s instructions using a cytotoxicity detection kit (Roche Diagnostic SpA., Milano, Italy). Tryptophan Fluorescence Measurements. Emission spectra of gBh (4 μM) containing tryptophan residues in the absence or presence of increasing quantities of shorter helical peptides (0.2, 2, 4 μM) were recorded between 310 and 400 nm with an excitation wavelength of 295 nm. The degree of peptide association was measured by the fluorescence intensity change as a function of the shorter peptide concentration, in three to four separate experiments. The fluorescence values were corrected by taking into account the dilution factor corresponding to the addition of microliter amounts of peptides and by subtracting the corresponding blank. Circular Dichroism Spectroscopy. CD spectra were recorded using a Jasco J-715 spectropolarimeter in a 1.0 cm quartz cell at room temperature. The spectra are an average of three consecutive scans from 260 to 195 nm, recorded with a bandwidth of 3 nm, a time constant of 16 s, and a scan rate of 10 nm/min. Spectra were recorded and corrected for the blank. Mean ellipticities (ME) were calculated using the expression ME = obsd/lc, where obsd is the ellipticity measured in millidegrees, l is the path length of the cell in cm, and c is the peptide concentration in mol/L. Solutions of peptides (20 μM) were prepared in 5 mM phosphate buffer pH 7.4 and in various percentages of TFE. The CD spectra of the most active peptide gBh1m (1, 10, and 20 μM) were also obtained at pH 7.4, 6.5, and 5.5. Mixing experiments were performed by comparing the spectrum of the two peptides mixed together at a total concentration of 40 μM (experimental spectrum) with the sum of the individual spectra of the peptides at a concentration of 20 μM (theoretical spectrum) in 5 mM phosphate buffer pH 7.4. Surface Plasmon Resonance. SPR experiments were carried out with a BIAcore 3000 analytical system (BIAcore, Uppsala, Sweden) using a streptavidin-coated (SA) sensor chip at 25 °C. The SA chip was purchased from BIAcore AB (Sweden); the SA molecules were preimmobilized on a 50 nm CM3 dextran matrix through amine coupling by the manufacturer. Before use, the chips were activated with three consecutive 1 min injections of 50 mM NaOH in 1 M NaCl, in accordance with the manufacturer’s instructions. The running buffer used for all experiments was PBS (pH 7.4); all solutions were freshly prepared, degassed, and filtered through 0.22 μm pores. The operating temperature was 25 °C. After cleaning as indicated by the manufacturers, the BIAcore instrument continued to run overnight using Milli-Q water as eluent to thoroughly wash all liquid-handling parts of the instrument. At first, biotinylated gBh (140 μL, 50 nM) was applied to the chip surface at a flow rate of 2 μL/min, while we used a biotinylated scrambled peptide for the reference channel. Second, a solution of gBh1m (140 μL at a flow rate of 20 μL/min) was injected onto the surface in the running buffer. Analysis of the gBh/gBh1m binding event was performed from a series of sensorgrams collected at different peptide concentrations (200 nM, 500 nM, 700 nM, 800 nM, 1 μM). The sensorgrams for each interaction were analyzed by curve fitting using numerical integration analysis. The BIAevaluation analysis package (version 4.1, GE Healthcare, Milan, Italy) was used to subtract the reference channel signal and to evaluate KD values. Several curve fitting algorithms were used, but good fit was obtained only with the two-state reaction model. Molecular Dynamics Simulations. MD simulations were conducted on a model generated from the crystal structure of extracellular domain of glycoprotein B from herpes simplex virus type I (PDB code 2GUM).21 In particular, the simulated model contained

avoided during transport of the protein in infected cells; such as the use of synthetic peptides mimicking the part of the long helix that is already exposed in the prefusion structure. Moreover, pH-dependent conformational changes could be correlated with the endosomal entry but not during entry at the plasma membrane or during cell−cell spread. However, if the conformational changes from the prefusion to the postfusion conformation are necessary, then another yet-unknown viral or host accessory protein is involved in triggering the same conformational change during plasma membrane entry. Our results demonstrate that peptides corresponding to the helical sequence of gB are attractive for further clinical development and provide further understanding of the complex mechanism of HSV-1 viral fusion, demonstrating that the scheme established for VSV G protein may represent a paradigm for the complex structural transitions of other class III fusion proteins.

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EXPERIMENTAL SECTION

Materials. Fluorenylmethoxycarbonyl (Fmoc)-protected amino acids were from INBIOS (Pozzuoli, NA, Italy), and NovaSyn TGA resin was from Nova Biochem (Darmstadt, Germany). The reagents for solid-phase peptide synthesis (piperidine, pyridine) were from Fluka (Sigma-Aldrich, Milano, Italy); trifluoroacetic acid (TFA) and acetic anhydride were from Applied Biosystems (Foster City, CA). H2O, N,N-dimethylformamide (DMF), and acetonitrile (CH3CN) were from LAB-SCAN (Dublin, Ireland). Dichloromethane (DCM) and methanol, HPLC-grade solvents, were from Merck (Darmstadt, Germany). Biotin-COOH was from Sigma (St. Louis, MO). Peptide Synthesis. Peptides were synthesized using the standard solid-phase 9-fluorenylmethoxycarbonyl (Fmoc) method as previously reported.13 All purified peptides were obtained in good yields (30− 40%). Table 1 shows the sequences of all synthesized peptides. After Fmoc deprotection of the last amino acid on the peptidyl resin, biotin was coupled (3-fold excess). The coupling reaction was repeated twice, for 2 h each time. At the end of the reaction, the biotinylated peptide was cleaved and purified as previously reported.13 In particular, peptides were purified by preparative RP-HPLC using a gradient of acetonitrile (0.1% TFA) in water (0.1% TFA) from 5% to 70% over 20 min. Peptide purity was confirmed by LC-MS. All purified peptides were obtained in a purity of ≥95%. Virus Entry Assays. Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. HSV-1, carrying a LacZ gene driven by the CMV IE-1 promoter to express β-galactosidase, was propagated as previously described.11 BoHV-1 (Cooper strain; ATCC VR-864) was replicated in MadinDarby bovine kidney (MDBK; ATCC CCL22) cells in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) at 35 °C in a 5% CO2 humidified atmosphere. Peptides were dissolved in DMEM without serum and used at a range of concentrations. All experiments were conducted in parallel with a scrambled peptide and no-peptide controls as reported previously.58 To assess the effect of peptides on inhibition of HSV infectivity, three different ways of treating cell monolayers were performed: (a) For “virus pretreatment”, approximately 2 × 104 PFU of virus was incubated in the presence of different concentrations of peptides (10, 50, 100, 200 μM) for 45 min at 37 °C and subsequently titrated on cell monolayers. (b) For “cotreatment”, the cells were incubated with increasing concentrations of the peptides (10, 50, 100, 200 μM) in the presence of serial dilutions of viral inoculum for 45 min at 37 °C. (c) For “post-treatment”, cell monolayers were infected with virus for 45 min at 37 °C. A range of concentrations of peptides (10, 50, 100, 200 μM) was then added to the inoculum, followed by a further 30 min incubation at 37 °C. 8373



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residues 112−153 (parts of domains IV, III, II), 365−459 (domain II), and 492−575 (domain III). The resulting model was optimized with a series of energy minimizations both in vacuo and in water. Parallel MD simulations (100 ns each) were performed with the GROMACS package by using the OPLS and AMBER force fields with TIP4Pew explicit water models. The simulations were carried out in the NPT ensemble with periodic boundary conditions at a constant temperature of 300 K. A rectangular box has been employed for accommodating the protein/peptide, water molecules, and ions. The Berendsen algorithm has been applied for the temperature and pressure coupling. The bonds were constrained by the LINCS algorithm. The particle-mesh Ewald (PME) method was used to account for the electrostatic contribution to nonbonded interactions (grid spacing of 0.12 nm). To ensure a system at pH 7, the protonation states of pH-sensitive residues were as follows: Arg and Lys were positively charged, Asp and Glu were negatively charged, and His was neutral. The net charge of the protein was neutralized by the addition of Cl− ions. The structural stability in the simulations was checked using routines implemented in GROMACS. The model of “open gB” in Figure 5 was obtained starting from a representative bent structure of the AMBER MD trajectory and upon superposition of domain II and domain I of the gB crystallographic structure on domain II of the simulated model.

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(3) Delboy, M. G.; Patterson, J. L.; Hollander, A. M.; Nicola, A. V. Nectin-2-mediated entry of a syncytial strain of herpes simplex virus via pH-independent fusion with the plasma membrane of Chinese hamster ovary cells. Virol. J. 2006, 3, 105. (4) Roller, D. G.; Dollery, S. J.; Doyle, J. L.; Nicola, A. V. Structurefunction analysis of herpes simplex virus glycoprotein B with fusionfrom-without activity. Virology 2008, 382 (2), 207−216. (5) Arii, J.; Uema, M.; Morimoto, T.; Sagara, H.; Akashi, H.; Ono, E.; Arase, H.; Kawaguchi, Y. Entry of herpes simplex virus 1 and other alphaherpesviruses via the paired immunoglobulin-like type 2 receptor alpha. J. Virol. 2009, 83 (9), 4520−4527. (6) Milne, R. S.; Nicola, A. V.; Whitbeck, J. C.; Eisenberg, R. J.; Cohen, G. H. Glycoprotein D receptor-dependent, low-pH-independent endocytic entry of herpes simplex virus type 1. J. Virol. 2005, 79 (11), 6655−6663. (7) Nicola, A. V.; Straus, S. E. Cellular and viral requirements for rapid endocytic entry of herpes simplex virus. J. Virol. 2004, 78 (14), 7508−7517. (8) Connolly, S. A.; Jackson, J. O.; Jardetzky, T. S.; Longnecker, R. Fusing structure and function: a structural view of the herpesvirus entry machinery. Nat. Rev. Microbiol. 2011, 9 (5), 369−381. (9) Galdiero, S.; Falanga, A.; Tarallo, R.; Russo, L.; Galdiero, E.; Cantisani, M.; Morelli, G.; Galdiero, M. Peptide inhibitors against herpes simplex virus infections. J. Pept. Sci. 2013, 19 (3), 148−158. (10) Turner, A.; Bruun, B.; Minson, T.; Browne, H. Glycoproteins gB, gD, and gHgL of herpes simplex virus type 1 are necessary and sufficient to mediate membrane fusion in a Cos cell transfection system. J. Virol. 1998, 72 (1), 873−875. (11) Galdiero, S.; Vitiello, M.; D’Isanto, M.; Falanga, A.; Collins, C.; Raieta, K.; Pedone, C.; Browne, H.; Galdiero, M. Analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type 1 glycoproteins H and B. J. Gen. Virol. 2006, 87 (Pt 5), 1085−1097. (12) Galdiero, S.; Falanga, A.; Vitiello, G.; Vitiello, M.; Pedone, C.; D’Errico, G.; Galdiero, M. Role of membranotropic sequences from herpes simplex virus type I glycoproteins B and H in the fusion process. Biochim. Biophys. Acta 2010, 1798 (3), 579−591. (13) Galdiero, S.; Falanga, A.; Vitiello, M.; Browne, H.; Pedone, C.; Galdiero, M. Fusogenic domains in herpes simplex virus type 1 glycoprotein H. J. Biol. Chem. 2005, 280 (31), 28632−28643. (14) Galdiero, S.; Falanga, A.; Vitiello, M.; D’Isanto, M.; Cantisani, M.; Kampanaraki, A.; Benedetti, E.; Browne, H.; Galdiero, M. Peptides containing membrane-interacting motifs inhibit herpes simplex virus type 1 infectivity. Peptides 2008, 29 (9), 1461−1471. (15) Galdiero, S.; Falanga, A.; Vitiello, M.; D’Isanto, M.; Collins, C.; Orrei, V.; Browne, H.; Pedone, C.; Galdiero, M. Evidence for a role of the membrane-proximal region of herpes simplex virus Type 1 glycoprotein H in membrane fusion and virus inhibition. ChemBioChem 2007, 8 (8), 885−895. (16) Galdiero, S.; Falanga, A.; Vitiello, M.; Raiola, L.; Fattorusso, R.; Browne, H.; Pedone, C.; Isernia, C.; Galdiero, M. Analysis of a membrane interacting region of herpes simplex virus type 1 glycoprotein H. J. Biol. Chem. 2008, 283 (44), 29993−30009. (17) Galdiero, S.; Falanga, A.; Vitiello, M.; Raiola, L.; Russo, L.; Pedone, C.; Isernia, C.; Galdiero, M. The presence of a single Nterminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type 1 glycoprotein H. J. Biol. Chem. 2010, 285 (22), 17123−17136. (18) Galdiero, S.; Russo, L.; Falanga, A.; Cantisani, M.; Vitiello, M.; Fattorusso, R.; Malgieri, G.; Galdiero, M.; Isernia, C. Structure and orientation of the gH625−644 membrane interacting region of herpes simplex virus type 1 in a membrane mimetic system. Biochemistry 2012, 51 (14), 3121−3128. (19) Chowdary, T. K.; Cairns, T. M.; Atanasiu, D.; Cohen, G. H.; Eisenberg, R. J.; Heldwein, E. E. Crystal structure of the conserved herpesvirus fusion regulator complex gH-gL. Nat. Struct. Mol. Biol. 2010, 17 (7), 882−888. (20) Matsuura, H.; Kirschner, A. N.; Longnecker, R.; Jardetzky, T. S. Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/

ASSOCIATED CONTENT

S Supporting Information *

Details of the MD study and cartoon representation of the gB postfusion structure highlighting the two patches of Glu residues. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +39 081 2534503; e-mail: stefania.galdiero@ unina.it. *Telephone: +39 081 5667646; e-mail: massimiliano.galdiero@ unina2.it. Author Contributions ▽

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by PON-1_2388 − Verso la medicina personalizzata: nuovi sistemi molecolari per la diagnosi e la terapia di patologie oncologiche ad alto impatto sociale and by the Italian MIUR - FIRB RBAP114AMK_006.

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ABBREVIATIONS USED HSV, herpes simplex virus; EBV, Epstein−Barr virus; TM, transmembrane; VSV, vesicular stomatitis virus; LDH assay, lactate dehydrogenase activity assay; TFE, trifluoroethanol; SPR, surface plasmon resonance; RU, response units; DSSP dictionary, dictionary of protein secondary structure; CH3CN, acetonitrile; DMEM, Dulbecco’s modified Eagle’s medium; MEM, minimum essential medium; FBS, fetal bovine serum; PFU, plaque-forming unit; CMC, carboxymethylcellulose

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REFERENCES

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simplex virus core fusion complex (gB-gH/gL) in an acidic environment. J. Virol. 2011, 85 (13), 6175−6184. (57) Dollery, S. J.; Wright, C. C.; Johnson, D. C.; Nicola, A. V. LowpH-dependent changes in the conformation and oligomeric state of the prefusion form of herpes simplex virus glycoprotein B are separable from fusion activity. J. Virol. 2011, 85 (19), 9964−9973. (58) Tarallo, R.; Carberry, T. P.; Falanga, A.; Vitiello, M.; Galdiero, S.; Galdiero, M.; Weck, M. Dendrimers functionalized with membrane-interacting peptides for viral inhibition. Int. J. Nanomed. 2013, 8, 521−534.

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