Current address: National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Klong Luang, Thailand
Current address: Program in Emerging Infectious Disease, Duke –NUS Graduate Medical School, and Department of Biological Sciences, National University of Singapore, Singapore, Singapore
Current address: Department of Microbiology, University of Washington School of Medicine, Seattle, Washington, United States of America
Current address: School of Public Health, University of Florida, Gainesville, Florida, United States of America
Conceived and designed the experiments: JMC EJ SI RS SFM. Performed the experiments: JMC EJ SML EH KAC KAF CRR. Analyzed the data: JMC EJ SML KAF MGR SI RS SFM. Contributed reagents/materials/analysis tools: JMC EJ SML MGR SI RS SFM. Wrote the paper: JMC SFM.
The authors acknowledge that Florida Gulf Coast University, Tulane University, and the University of Washington have submitted patent applications covering the peptides described in this manuscript.
Viral fusogenic envelope proteins are important targets for the development of inhibitors of viral entry. We report an approach for the computational design of peptide inhibitors of the dengue 2 virus (DENV-2) envelope (E) protein using high-resolution structural data from a pre-entry dimeric form of the protein. By using predictive strategies together with computational optimization of binding “pseudoenergies”, we were able to design multiple peptide sequences that showed low micromolar viral entry inhibitory activity. The two most active peptides, DN57opt and 1OAN1, were designed to displace regions in the domain II hinge, and the first domain I/domain II beta sheet connection, respectively, and show fifty percent inhibitory concentrations of 8 and 7 µM respectively in a focus forming unit assay. The antiviral peptides were shown to interfere with virus:cell binding, interact directly with the E proteins and also cause changes to the viral surface using biolayer interferometry and cryo-electron microscopy, respectively. These peptides may be useful for characterization of intermediate states in the membrane fusion process, investigation of DENV receptor molecules, and as lead compounds for drug discovery.
Virus surface proteins mediate interactions with target cells during the initial events in the process of infection. Inhibiting these proteins is therefore a major target for the development of antiviral drugs. However, there are a very large number of different viruses, each with their own distinct surface proteins and, with just a few exceptions, it is not clear how to build novel molecules to inhibit them. Here we applied a computational binding optimization strategy to an atomic resolution structure of dengue virus serotype 2 envelope protein to generate peptide sequences that should interact strongly with this protein. We picked dengue virus as a target because it is the causative agent for the most important mosquito transmitted viral disease. Out of a small number of candidates designed and tested, we identified two different highly inhibitory peptides. To verify our results, we showed that these peptides block virus:cell binding, interfere with a step during viral entry, alter the surface structure of dengue viral particles, and that they interact directly with dengue virus envelope protein. We expect that our approach may be generally applicable to other viral surface proteins where a high resolution structure is available.
Fusogenic viral envelope glycoproteins are multimeric proteins that facilitate the fusion of viral and target cell lipid membranes during the initiation of infection. The membrane fusion process is energetically favorable and essentially irreversible, but has a considerable kinetic energy barrier
There are several distinct types of viral fusion proteins, including the class I, primarily alpha helical proteins (such as HIV TM and influenza HA), the class II, primarily beta sheet proteins (such as the flavivirus E and alphavirus E1), and mixed helix/sheet proteins (including herpes virus gB and rhabdovirus G)
Only a few examples of viral entry inhibitors with activity against the primarily beta sheet envelope proteins (E) from flaviviruses have been described
The causative agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, DENV has emerged in the past several decades as the most important mosquito borne viral disease with an estimated 2.5 billion people living in areas at risk for epidemic transmission and 50–100 million people infected annually
Peptide inhibitors were designed to have improved in situ binding compared to naturally occurring sequences using the residue-specific all-atom probability discriminatory function (RAPDF)
A 20 residue acid sliding window that moved from the N to the C terminus of the E protein in 10 residue acid increments was evaluated by a structural stability (pseudoenergy) optimization protocol using the RAPDF. A Metropolis Monte Carlo search algorithm
DENV-2 strain NG-C was obtained from R. Tesh at the University of Texas at Galveston. Virus was propagated in the
Peptides were synthesized by solid-phase N-α-9-flurenylmethyloxycarbonyl chemistry, purified by reverse-phase high performance liquid chromatography and confirmed by amino acid analysis and electrospray mass spectrometry (Genemed Synthesis, San Antonio, TX). Peptide stock solutions were prepared in 20% (v/v) dimethyl sulfoxide (DMSO): 80% (v/v) H2O, and concentrations determined by absorbance of aromatic side chains at 280 nm.
LLC-MK2 target cells were seeded at a density of 1×105 cells in each well of a 6-well plate 24 h prior to infection. Approximately 200 focus forming units (FFU) of virus were incubated with or without peptide in serum-free DMEM for 1 h at rt. Virus/peptide or virus/control mixtures were allowed to infect confluent target cell monolayers for 1 h at 37°C, with rocking every 15 m, after which time the medium was aspirated and overlaid with fresh DMEM/10% (v/v) FBS containing 0.85% (w/v) Sea-Plaque Agarose (Cambrex Bio Science, Rockland, ME) without rinsing. Cells with agar overlays were incubated at 4°C for 20 m to set the agar. Infected cells were then incubated at 37°C with 5% CO2 for 5 days. Infected cultures were fixed with 10% formalin overnight at 4°C, permeablized with 70% (v/v) ethanol for 20 m, and rinsed with phosphate buffered saline, pH 7.4 (PBS) prior to immunostaining. Virus foci were detected using a specific mouse mAb from hybridoma E60 (obtained from M. Diamond at Washington University), followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (Pierce, Rockford, IL), and developed using AEC chromogen substrate (Dako, Carpinteria, CA). Results are expressed as the average of at least two independent trials with three replicates each. IC50 values were determined using variable slope sigmoidal dose-response curve fits with GraphPad Prism 4.0 software (LaJolla, CA), except for DN81opt, which was determined graphically due to a lack of data points to produce a reasonable curve fit.
Cytotoxicity of peptides was measured by monitoring mitochondrial reductase activity using the TACS™ MTT cell proliferation assay (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. Dilutions of peptides in serum-free DMEM were added to confluent monolayers of LLC-MK2 cells in 96-well plates for 1 h at 37°C, similar to the focus forming inhibition assays, and incubated at 37°C with 5% (v/v) CO2 for 24 h. Absorbance at 560 nm was measured using a Tecan GeniosPro plate reader (Tecan US, Durham, NC).
DENV-2 NGC strain used for the cryoEM reconstructions was propagated in mosquito C6/36 cells. Virus was purified by precipitation with 40% PEG 8000 and then ultracentrifugation onto a 25% sucrose cushion. Virus was further purified by banding on a 10%–30% potassium tartrate gradient. The virus band was removed and dialyzed against 12 mM Tris pH 8.0, 120 mM NaCl, 1 mM EDTA, and concentrated using a Millipore Centricon filter. Purified virus was mixed with 1OAN or DN57opt at a concentration of 1 molecule of peptide for every E protein on the surface of the virus. The complex was incubated for half an hour at 37°C followed by half an hour at 4°C and then flash frozen on holey carbon grids in liquid ethane. Images of the frozen complex were taken with a Philips CM200 FEG transmission electron microscope (Philips, Eindhoven, The Netherlands) at a magnification 51,040 using an electron dose of approximately, 25e-/Å 2 using a Charge-Couple device.
Real time binding assays between peptides and purified DENV-2 S1 E protein were performed using biolayer interferometry on an Octet QK system (Fortebio, Menlo Park, CA). This system monitors interference of light reflected from the surface of a fiber optic sensor to measure the thickness of molecules bound to the sensor surface. Purified, recombinant, 80% truncated DENV-2 S1 E protein was obtained from Hawaii Biotechnology (Honolulu, HI). Peptides were N-terminally biotinylated with a 5∶1 molar ratio of NHS-LC-LC-Biotin (Pierce/ThermoFisher, Rockford, IL) in PBS pH 6.5 at 4°C. Excess biotinylation reagent was removed using Pepclean C-18 spin columns (Pierce/ThermoFisher, Rockford, IL). Biotinylated peptides were coupled to kinetics grade streptavidin high binding biosensors (Fortebio, Menlo Park, CA) at several different concentrations. Sensors coated with peptides were allowed to bind to E protein in PBS with 0.02% (v/v) Tween-20 and 1 mg/ml BSA at several different E protein concentrations. Binding kinetics were calculated using the Octet QK software package, which fit the observed binding curves to a 1∶1 binding model to calculate the association rate constants. E protein was allowed to dissociate by incubation of the sensors in PBS. Dissociation curves were fit to a 1∶1 model to calculate the dissociation rate constants. Binding affinities were calculated as the kinetic dissociation rate constant divided by the kinetic association rate constant.
Approximately 200 FFU of DENV-2 without peptide was allowed to bind and enter target cells for 1 h at 37°C as described for the focus forming unit assay. Unbound virus was then removed by rinsing with PBS and peptide was added to the cells for 1 h at 37°C. Cultures were washed again in PBS and agarose overlays, incubation, and immunological detection was conducted as described for the focus forming unit assay.
Approximately 200 FFU of DENV-2 were allowed to attach to cells for 45 min at 4°C, and then rinsed with cold PBS before peptide was incubated with the target cells for 45 min at 4°C. The cells were rinsed again with cold PBS, and agarose overlays, incubation, and immunological detection were conducted as described for the focus forming unit assay.
Hemagglutination inhibition (HI) was performed according to
Binding inhibition assays were modified from Thaisomboonsuk, et al
Graphs were generated using KaleidaGraph v.3.6 graphing software (Synergy Software, Reading, PA). Statistical analyses were performed using the GraphPad Prism 4.0 software package (GraphPad Software, San Diego, CA). P values less than 0.05 were considered significant.
We had previously identified several E protein regions where peptides mimicking the E protein sequence might function as inhibitors. Several of these mimic peptides did not show substantial DENV inhibitory activity
Name | LOCATION | Sequence | IC50 (µM) |
DN57wt | 205–232 | AWLVHTQWFLDLPLPWLPGADTQGSNWI | --* |
DN57opt | RWMVWRHWFHRLRLPYNPGKNKQNQQWP | 8±1 | |
DN57opt-scram | RWRHLKKMQRLQPRNPNWPGQFWVHYNW | -- | |
DN80wt | 96–114 | MVDRGWGNHAGLFGKGSIV | --* |
DN80opt | MVIVQHQWMQIMRWPWQPE | -- | |
DN81wt | 205–223 | AWLVHRQWFLDLPLPWLPG | --* |
DN81opt | RQMRAWGQDYQHGGMGYSC | 36±6 | |
1OAN1wt | 41–60 | LDFELIKTEAKQPATLRKYC | ND |
1OAN1 | FWFTLIKTQAKQPARYRRFC | 7±4 | |
1OAN1-scram | QQCFRFPALRKKATYTRFWI | -- | |
1OAN2wt | 131–150 | QPENLEYTVVITPHSGEEHA | ND |
1OAN2 | YPENLEYRVYITPHPGEEHH | -- | |
1OAN3wt | 251–270 | VVLGSQEGAMHTALTGATEI | ND |
1OAN3 | EWSKHREGRWHTALTGATEI | -- | |
1OAN4wt | 351–370 | LITVNPIVTEKDSPVNIEAE | ND |
1OAN4 | WHTVEPIVTEKDRPVNYEWE | -- |
Names and sequences for previously tested wild type peptides are denoted with an asterisk
To identify additional novel peptide inhibitors and their corresponding targets, a 20 residue sliding window that moved from the N to the C terminus of the DENV-2 strain S1 E protein (PDB ID 1OAN) in 10 residue acid increments was evaluated by a structural stability (pseudoenergy) optimization protocol using the RAPDF. A Metropolis Monte Carlo search algorithm
(A) The DENV-2 E protein is shown linearly from N to C terminus. The three domains are color coded above, domain I is shown in red, domain II is yellow, and domain III is in blue according to
In order to verify the effectiveness of the binding optimization process and peptide design, synthesized peptides were tested for antiviral activity against DENV-2 strain NG-C in a focus forming unit (FFU) reduction assay. DENV-2 strains S1 (GenBank accession number M19197.1) and NG-C (GenBank accession number AF038403.1) share 98% amino acid sequence identity in the E protein and the majority of differences are conservative. Dose response curves generated for the optimized peptides DN57opt, DN80opt, and DN81opt are shown in
Increasing concentrations of optimized inhibitor peptides and corresponding scrambled peptides of identical composition were tested against DENV-2 in a focus forming unit reduction assay. (A) Optimized peptides (B) DN57opt and corresponding scrambled peptide of identical composition (C) Novel peptides (D) 1OAN1 and corresponding scrambled peptide of identical composition. Error bars are ±sem.
Because toxicity could result in a decrease in focus formation and be interpreted as evidence of antiviral activity, the inhibitory peptides and their scrambled versions were assessed for cellular toxicity. Confluent monolayers of LLC-MK2 cells used in FFU reduction assays were exposed to increasing concentrations of peptide before measuring mitochondrial reductase activity using an MTT mitochondrial reductase activity assay (
Increasing concentrations of peptides were tested in an MTT mitochondrial reductase activity assay. Error bars are ± sd. (A) DN57opt (B) Scrambled version of DN57opt (C) 1OAN1 (D) Scrambled version of 1OAN1. * denotes a statistically significant difference from the no peptide control.
Cryoelectron microscopy (cryoEM) was used to visualize the effect of the DN57opt and 1OAN1 peptides on DENV-2 viral particles. Control dengue virions exhibited the normal, nearly smooth outer surface typical of mature flaviviruses
Purified and concentrated virus was prepared with or without incubation with peptides and then flash frozen for imaging. Panels show (A) virus only, (B) virus incubated with DN57opt, (C) virus incubated with 1OAN1. Scale bars indicate 100 nm.
Biolayer interferometry was performed to examine binding of the peptides to purified, truncated DENV-2 E protein. Amino terminally biotinylated peptides were immobilized onto streptavidin biosensors and then the association and dissociation of truncated E protein with the immobilized peptides was monitored. The interactions of three different concentrations of truncated E protein to peptides DN57opt and 1OAN1 are shown (
Biolayer interferometry was used to assay the binding of the peptides to truncated E Protein. The association and dissociation of increasing concentrations of truncated E protein to peptides DN57opt (A) and 1OAN1 (B) are shown. A buffer blank (PBS, 0.02% Tween-20, 0.1% BSA) containing no E protein was run for each peptide. The affinity of the peptides for the truncated E protein was calculated (DN57opt KD = 1.2×10−6±0.6×10−6 M (mean±sd), 1OAN1 KD = 4.5×10−7±2.0×10−7 M).
In order to determine if the peptides were exerting their effects on post-entry steps in the virus replication cycle, DENV-2 was allowed to infect LLC-MK2 cells before peptide was added to the cells (
Treatment of cells with increasing concentrations of (A) DN57opt and (B) 1OAN1 after DENV-2 has infected cells shows no significant inhibition. Treatment with (C) DN57opt or (D) 1OAN1 after DENV-2 has bound to LLCMK-2 cells at 4°C for one hour inhibits infection. Error bars are ±sem.
Since we had determined that inhibition with both peptides occurs at a viral entry step, we asked if infection could still be inhibited after virus had bound to the surface of target cells. We bound virus to cells at 4°C, then treated with increasing concentrations of DN57opt or 1OAN1 before warming the cells back to 37°C and allowing the infections to progress (
To determine if the peptides interefere with virus:cell interactions, we conducted two different experiments. We first performed hemagglutination inhibition assays, but were unable to detect any inhibition of the ability of viral antigen to agglutinate red blood cells (data not shown). To further investigate virus:cell binding in a more relevant system, we treated virus with DN57opt or 1OAN1, bound the virus to cells, and washed the cells repeatedly at 4°C before measuring the amount of virus remaining on the cells by quantitative rt-PCR. Both peptides showed evidence of ability to block virus:cell binding compared to control virus without peptide (
Virus pre-incubated with either DN57opt or 1OAN1 shows reduced binding to cells compared to control virus without peptide. Pre-incubation of virus with pooled human anti-dengue serum or heparan sulfate similarly shows reduced cell binding. * Indicates a significant difference (p<0.05) from all others by 1-way ANOVA followed by Tukey's posthoc test.
We have used computational methods to design multiple peptide inhibitors of the DENV E glycoprotein. Importantly, out of seven peptides synthesized and tested, two peptides with high activity and one peptide with intermediate activity were identified. A high resolution crystal structure of the pre-fusion conformation of the DENV-2 E
Neither peptide showed inhibitory activity when added directly to cells after infection had already occurred, indicating that the peptides were acting during an entry step in the virus life cycle, and sequence scrambled versions of the two most active peptides were inactive, confirming sequence specific activity. Both peptides also block virus:cell binding, but are still capable of inhibiting infection even when added after virions have already bound to the surface of target cells.
CryoEM was used to visualize the effect of the peptides on DENV-2 virions. The surface of virions appeared to change from smooth to rough after incubation with the antiviral peptides. This suggests that there may be an alteration of the arrangement of the surface envelope protein (
The DN57opt and 1OAN1 peptides were designed for optimized binding to the pre-fusion E structure and we show direct evidence for this interaction, both with the purified, monomeric E protein, and with virion particles. These peptides likely function by displacing portions of the E protein and interfering with normal cell binding or the structural changes during entry. Although separate in the primary protein sequence, the regions targeted in the design the DN57 and 1OAN1 peptides are partially adjacent to each other in the crystal structure, with the C terminus of the 1OAN1 region occupying a pocket surrounded by the DN57 region (See
Despite difficulties with oral administration and degradation in the digestive tract, peptides may make useful antiviral agents when targeted against viral envelope proteins. Directing inhibitors to viral surface proteins avoids the major difficulty of crossing cellular membranes in order to reach the target. For example, peptide inhibitors of intercellular viral targets, such as proteases or polymerases, would need to cross the cell plasma membrane, and in the case of flaviviruses, possibly internal membrane bound replication and assembly compartments. The HIV entry inhibitor T-20 (Fuzeon) is a peptide, and in the context of a chronic infection, repeated life-long injections are problematic. DENV is an acute infection and most severe DENV infections require intravenous fluid support, facilitating delivery of anti-DENV peptides by this route.
We have established the existence of multiple, distinct inhibitory peptides targeting the DENV E glycoprotein and confirmed the utility of rational design using structural data for developing DENV E protein inhibitors. Applications of this strategy should also be possible for the generation and refinement of lead compounds for other viral envelope fusion proteins. It would be optimistic to propose that any single antiviral would provide an effective treatment for DENV given the enormous genetic variability of the four serotypes and multiple substrains. Different classes of inhibitors targeting the E protein and other DENV targets
Translation of the abstract into Thai by Ekachai Jenwitheesuk.
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Translation of the abstract into Spanish by Sharon Isern.
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