Model Membrane Platforms for Biomedicine: Case Study on Antiviral Drug Development
© The Author(s) 2012
Received: 19 September 2011
Accepted: 29 December 2011
Published: 11 February 2012
As one of the most important interfaces in cellular systems, biological membranes have essential functions in many activities such as cellular protection and signaling. Beyond their direct functions, they also serve as scaffolds to support the association of proteins involved in structural support, adhesion, and transport. Unfortunately, biological processes sometimes malfunction and require therapeutic intervention. For those processes which occur within or upon membranes, it is oftentimes difficult to study the mechanism in a biologically relevant, membranous environment. Therefore, the identification of direct therapeutic targets is challenging. In order to overcome this barrier, engineering strategies offer a new approach to interrogate biological activities at membrane interfaces by analyzing them through the principles of the interfacial sciences. Since membranes are complex biological interfaces, the development of simplified model systems which mimic important properties of membranes can enable fundamental characterization of interaction parameters for such processes. We have selected the hepatitis C virus (HCV) as a model viral pathogen to demonstrate how model membrane platforms can aid antiviral drug discovery and development. Responsible for generating the genomic diversity that makes treating HCV infection so difficult, viral replication represents an ideal step in the virus life cycle for therapeutic intervention. To target HCV genome replication, the interaction of viral proteins with model membrane platforms has served as a useful strategy for target identification and characterization. In this review article, we demonstrate how engineering approaches have led to the discovery of a new functional activity encoded within the HCV nonstructural 5A protein. Specifically, its N-terminal amphipathic, α-helix (AH) can rupture lipid vesicles in a size-dependent manner. While this activity has a number of exciting biotechnology and biomedical applications, arguably the most promising one is in antiviral medicine. Based on the similarities between lipid vesicles and the lipid envelopes of virus particles, experimental findings from model membrane platforms led to the prediction that a range of medically important viruses might be susceptible to rupturing treatment with synthetic AH peptide. This hypothesis was tested and validated by molecular virology studies. Broad-spectrum antiviral activity of the AH peptide has been identified against HCV, HIV, herpes simplex virus, and dengue virus, and many more deadly pathogens. As a result, the AH peptide is the first in class of broad-spectrum, lipid envelope-rupturing antiviral agents, and has entered the drug pipeline. In summary, engineering strategies break down complex biological systems into simplified biomimetic models that recapitulate the most important parameters. This approach is particularly advantageous for membrane-associated biological processes because model membrane platforms provide more direct characterization of target interactions than is possible with other methods. Consequently, model membrane platforms hold great promise for solving important biomedical problems and speeding up the translation of biological knowledge into clinical applications.
Biological membranes represent one of the most important interfaces in cellular systems [1–3]. From serving as a physical barrier  to regulating signal transduction pathways , they play a key role in cellular protection and homeostasis. Beyond their own direct functions, membranes are also scaffolds upon which proteins associate to stabilize cellular structure , promote adhesion , and direct transport processes , among other functions. As one measure of the membrane interface’s biomedical importance, around 60% of approved drug targets are membrane-associated proteins . In addition, many viral pathogens take advantage of membranes as part of their life cycle. Specifically, membranes serve as a platform for viral proteins to bind to the host cell  as well as to organize themselves into complexes that catalyze critical synthesis activities during viral genome replication .
Given all of these events that take place at the membrane interface, there are many possibilities for therapeutic intervention to target disease-related processes such as abnormal signaling , viral membrane fusion , and inflammatory cascades . Beyond the wide range of membrane-associated processes amenable to molecular targeting, there can also be multiple strategies against one target. For example, ion channels embedded in lipid membranes may either be stimulated or inhibited by electrical force, mechanical force, or chemical cues . Other therapeutic possibilities include targeting the lipid membrane to modulate its physical properties including permeability , as well as disrupting the enzymatic activities of membrane-associated proteins  or their interactions with other macromolecule components . Regardless of the biomedical problem, it is necessary to understand the fundamental molecular mechanisms of the target interaction.
Continuing progress in the development of biological tools has led to major advances for determining the structure and function of biological macromolecules. Biophysical methods such as nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy have enabled structural characterization of proteins in their membrane-associated states [18, 19]. This information has significantly improved fundamental understanding of the structure–function relationships of membrane-associated proteins, as well as aid in the identification of new therapeutic targets. However, to date, a major challenge has been monitoring the dynamics of membrane-protein interactions as well as protein–protein interactions and enzymatic processes at membrane interfaces.
To overcome this challenge, a common tactic has been to design soluble forms of analyte proteins that can be studied directly in solution , or in the presence of membrane-mimicking components such as detergents . Although the enzymatic activities of these recombinant proteins can typically be monitored [22, 23], no information is gained about the interaction of the protein with its host membrane environment, how this interaction affects its function, or how the step of membrane association may be targeted. As such, the amount of information that can be gained about the target interaction is limited when using non-native systems.
Engineering strategies offer a promising approach to study the interfacial science principles of membrane-associated biological activities in physiologically relevant settings. A central theme in the engineering sciences is the fundamental characterization of a model biological system by defining the necessary parameters and then systematically evaluating them. Consequently, this approach can help to understand the self-assembly and functional activity of biological systems by easily changing parameters. To facilitate characterization work, model systems are often studied with surface-sensitive analytical tools . A range of techniques exist to probe biological thin film interfaces, and can measure acoustic , optical , spectroscopic , electrical , chemical , and morphological properties . With this wealth of options, there are many analytical strategies to choose from in order to characterize the specific target interaction.
Regardless of the characterization methods, it is important to design an appropriate model system that mimics important structural properties of the target interaction [24, 31–33]. Importantly, for the study of membrane-associated biological processes, model membranes enjoy the advantage of self-assembly formation [31, 34], which permits facile adjustment of system parameters. As such, bottom-up design has enabled the development of model membrane systems for probing a wide range of biological processes such as cell adhesion, membrane fusion, and cellular signaling, and there are many excellent reviews already in the field [35–39]. However, there has been limited demonstration thus far of how knowledge gained from these approaches can be translated into clinically impactful biomedical applications. In this review article, we establish a framework to demonstrate how engineering strategies can leverage the sensing advantages of model membrane systems to yield novel therapies that are poised to enter the drug pipeline and solve challenging biomedical problems.
One of the most serious global health problems is the urgent need to combat infectious diseases. With the emergence of new infectious diseases as well as the reemergence of others, there are a number of socioeconomic, evolutionary, and environmental, and molecular biology issues which hinder the development of more effective diagnostic and therapeutic options. Indeed, one of the biggest hurdles is the need to identify new molecular targets in order to counter the emergence of drug-resistant pathogens. As a case study example of a viral pathogen target, we have selected the hepatitis C virus (HCV) and discuss how engineering strategies identified a previously unknown functional activity encoded within the HCV nonstructural 5A (NS5A) protein. By employing a model membrane platform to mimic the host cell membrane interface upon which viral genome replication occurs, a striking ability to lyse lipid vesicles was discovered within the NS5A N-terminal amphipathic, α-helix (AH) [40–43].
Because lipid vesicles also serve as a simplified model for the lipid envelope of virus particles, mechanistic studies predicted that a range of medically important viruses might be susceptible to rupturing treatment with a synthetic AH peptide . Importantly, these predictions were tested and validated by molecular virology experiments which demonstrated AH peptide has broad-spectrum antiviral activity against HCV, HIV, herpes simplex virus, and dengue virus, as well as many more deadly pathogens [44–46]. As such, the AH peptide represents the first in class of lipid envelope-rupturing antiviral agents. Beyond the identification of this one drug candidate, these studies have also led to the development of a new testing platform to rapidly characterize antiviral drug candidates in minutes. Taken together, model membrane technology demonstrates significant potential for biomedicine, and is yielding new biotechnology and clinical applications to solve a range of important biomedical problems.
2 Challenges of Hepatitis C Drug Development
Thus far, the principal antiviral therapeutic strategy has been vaccination . While this strategy has proven effective for eradicating viral pathogens such as polio and smallpox, vaccine development for several important viral diseases including respiratory tract viruses, human papilloma viruses, herpes viruses, and haemorrhagic fever viruses has been unsuccessful . Moreover, while vaccines are available for influenza virus types A and B and for Hepatitis B virus, effective treatment requires additional antiviral drugs, which are still prone to the emergence of viral resistance as well as variable tolerance and efficacies . Most notably, there is no promising vaccine in the pipeline for either HCV or HIV. Nonetheless, these viral pathogens are two of the deadliest. Over 40 million people worldwide are infected with HIV, while an astounding 170 million people are infected with HCV . Given the large-scale impact of these viral pathogens, we have selected HCV as a prototype case to demonstrate how model membranes offer a platform to target membrane-associated steps in the virus life cycle in order to catalyze the development of more powerful antiviral drugs.
Functional targeting of the HCV replicase
Membrane associating region
Necessary for replication
At least one transmembrane domain
(1) NS2–NS3 proteolytic cleavage
(1) Transmembrane domains
(2) Protease active site
(3) Dimerization motif
(1) Serine protease domain releases NS proteins downstream
(1) Protease active site
(2) Helicase active site
(2) Helicase domain unwinds dsRNA/ssRNA
(3) NTPase active site
(4) RNA-binding region
(5) NS4A- and NS5B-interaction regions
N-terminal transmembrane domain
(1) NS3 cofactor for proper folding and membrane localization
(1) N-terminal transmembrane domain
(1) Induces membranous web formation
(1) N-terminal AH
(2) Internal AH
(3) RNA-binding region
(4) Oligomerization motif
(1) N-terminal AH
(2) Domain I dimerization motif for RNA binding
(3) Core protein interaction region
C-terminal anchor region
(1) Synthesis of viral genome RNA
(1) Polymerase active site
(2) Allosteric binding sites
(3) NS3- and NS5A-interaction regions
(4) RNA-binding region
(5) Oligomerization motif
Like other positive-strand RNA viruses, HCV genome replication occurs in close association with host cell-derived membranes, particularly from the endoplasmic reticulum (ER). While genome replication of some viruses occurs on the surface of preexisting vesicular membranes , other viruses, including HCV, promote the formation of de novo membrane structures [55, 56]. Most of the HCV NS proteins interact together on a membranous platform (termed the “membranous web”) to form the replicase, a multi-protein complex that directs RNA polymerase activity [55, 57]. As inhibitors of the NS3 protease and NS5B polymerase are entering late-stage clinical trials  (see Table 1), it is already becoming clear that additional targets in the HCV replicase need to be identified to minimize the effects of viral resistance on successful clinical treatment . Therefore, a major area of research in the molecular virology of HCV is the identification of new targets for pharmacologic inhibition.
In recent years, significant focus has been directed at the HCV NS5A protein. While this protein is implicated in regulating host cell protein activities and modulating virus sensitivity to interferon treatment, it has no known enzymatic function [60–62]. Nevertheless, NS5A is a key component of the HCV replicase complex and is necessary for viral replication . Given that HCV replicase activity occurs at the membrane interface, it is interesting that NS5A possesses an N-terminal amphipathic, α-helix (AH) which is necessary for proper cytoplasmic membrane localization of NS5A in the host cell . Indeed, genetic mutation analysis confirmed that the NS5A AH is necessary for HCV replication . Together, the necessity of NS5A AH for membrane association and viral genome replication suggests that targeting membrane association of NS5A would represent a prime therapeutic target to inhibit replication . Compared to other HCV targets that are prone to viral resistance, the NS5A AH possesses significant sequence homology across all HCV isolates identified to date . Thus, its amphipathic, membrane-associating property is a conserved functional target. The design of a model system to investigate membrane association of the NS5A AH might lead to a viable inhibition strategy.
3 Engineering Strategy to Mimic Biological Membranes
Model membranes are simplified versions of cellular membranes that are based on lipid bilayer assemblies . For biological studies, the design emphasis is on mimicking the most fundamental properties of membranes including bilayer environment [34, 68] and fluidity  in order to successfully host membrane-associating motifs. There are two main types of model membranes: (1) intact vesicle platform (Fig. 3b); and (2) planar lipid bilayer (Fig. 3c). Although they can consist of the same lipid material, the physical properties of the planar bilayer and intact vesicle platforms are quite different.
The planar bilayer presents a flat, uniform surface with its lipid components exhibiting long-range, two-dimensional lateral diffusion . It can self-assemble on certain substrates such as mica and silicon oxide by the surface-induced vesicle fusion mechanism—i.e., adsorption and subsequent rupture of lipid vesicles after reaching a critical vesicle concentration on the substrate . Importantly, the platform is intimately associated with the supporting substrate. Collectively, these features have two effects. First, the planar bilayer is rigidly coupled to the substrate and therefore a thin hydration layer exists between the substrate and lower leaflet [34, 71]. Second, the surface pressure of the bilayer increases when peripheral membrane proteins such as phospholipase A2 adsorb to and partially insert themselves into the lipid membrane . This rise in surface pressure can terminate macromolecule adsorption at a lower binding saturation value than that of other model or biological systems .
In contrast, the intact vesicle platform is comprised of adsorbed, unruptured lipid vesicles. When spherical lipid vesicles adsorb to substrates such as gold and titanium oxide, they deform but do not rupture . The result is a layer of pancake-like vesicles whose morphology is non-uniform; the intrinsic curvature of this platform surface is important for detecting curvature-sensitive binding of proteins such as those containing BAR domains . The intact vesicle contains hydrodynamically coupled solvent that is encapsulated within its inner core and therefore the layer has viscoelastic properties [75, 76]. As a result, the layer is a soft film that is responsive to the binding of amphipathic macromolecules. Specifically, vesicles can modulate their physical properties in order to maintain a constant surface pressure during the course of a binding interaction . This flexibility enables the determination of binding saturation values that are more representative of biological systems.
In addition to model membranes, sensing platforms can consist of cell-derived membranes that contain a natural assortment of components including lipids, proteins, and glycosaminoglycans [77–79]. While model membranes represent a general platform to study membrane association processes driven by amphiphilic interactions, cell-derived membranes contain receptors which can mediate specific binding of proteins. When studied in conjunction, model and cell-derived membranes can identify the determinants of membrane association for a target protein. Indeed, this approach has significantly aided characterization of the binding dynamics of the NS5A AH in one recent study .
Cell-derived membranes from Huh7 cells—a human liver tumor cell line commonly used for HCV replication studies—were assembled on the sensor chip as platforms to probe binding of AH peptide. In this case, significantly more AH peptide associated to the cell-derived membrane as compared to the planar bilayer (Fig. 4c). However, NH peptide once again did not bind to the cell-derived platform (Fig. 4d). These comparison studies with the model and cell-derived membranes suggested that there is a specific binding receptor of the NS5A AH within Huh7 cell-derived membranes.
To confirm this binding specificity, the authors combined biochemical methods with the QCM-D platform. Specifically, pretreatment with trypsin removed proteinaceous components from the Huh7 cell membranes before experiment. When the interactions of AH peptide and transactivator of transcription (TAT)-derived peptide to these trypsin-treated membranes were monitored, only the TAT-derived peptide demonstrated binding (Fig. 4e). The binding mass values were calculated from the QCM-D results and confirmed that only the TAT-derived peptide showed a positive binding mass on the treated membrane platform (Fig. 4f). This discrepancy was expected because the TAT-derived peptide is believed to bind to membranes via extracellular glycosaminoglycans , and not protein receptors. By contrast, the sensitivity of the AH peptide binding to trypsin treatment further supports that it specifically interacts with a protein receptor.
In addition to these experiments, one more set of controls was performed to verify the removal of proteinaceous components by trypsin treatment. Protein Tyrosine Phosphatase 1B (PTP1B) is a membrane protein found in Huh7-derived membranes. In the absence of trypsin treatment, antibody binding to PTP1B was observed (Fig. 4g). However, after trypsin treatment, no binding occurred (Fig. 4h). As such, it was verified that trypsin treatment is a valid method to remove proteinaceous components in this assay format. While QCM-D monitoring enabled sensitive detection of membrane-peptide interactions, drawing conclusions from the results of the study required integrated analysis of the biochemical properties of the biomembrane-on-a-chip platform.
Based on this collective set of experiments, the AH peptide demonstrated significantly greater binding to cell-derived versus model membranes. A battery of biochemical control experiments confirmed that this difference is caused by proteinaceous components only found in the cell-derived membranes. By combining the model and cell-derived membrane platforms, the authors were able to support but not confirm the hypothesis that a protein receptor for the NS5A AH resides in the membrane of the host cell endoplasmic reticulum.
4 Fundamental Characterization of Model Biological System
To further probe membrane association of the NS5A AH, there are several characterization strategies that can be taken based on the observation that proteinaceous components influence the binding of AH peptide to lipid membranes in one of two ways. First, the AH peptide ligand may directly bind a specific protein receptor of the host cell, as proposed in the initial hypothesis. Second, proteinaceous components may modulate the properties of the lipid membrane itself, thus affecting the binding interaction of the AH peptide to lipid and/or other protein components. Because the AH peptide is amphipathic, physical properties of the lipid membrane are likely to play a significant role in this interaction. While the AH peptide demonstrated minimal binding to a planar bilayer, it is important to determine whether this interaction kinetics holds for all types of model membranes or whether it is the result of the platform design. As previously discussed, there are other useful model membrane platforms to mimic cell membranes, including the intact vesicle platform which is a soft film that is more biologically relevant for studying macromolecular interaction kinetics.
Recent studies have investigated how AH peptide affects the film properties of the intact vesicle platform [40, 42]. With the collection of characterization data, there is an increasing amount of information about the molecular determinants of the target interaction. By understanding these parameters, the overall process of fundamental characterization can identify potential biomedical applications, and guide translational research directed at validating these applications. This work is an excellent example of how engineering strategies can reveal unexpected findings into the mechanisms of biological interactions.
4.1 Vesicle-to-Bilayer Structural Transformation by AH Peptide
While vesicle rupturing is typical of membrane lysis processes catalyzed by peptides [89, 90], this result is particularly exciting because it is the first example of a biological interaction that promotes the structural transformation of a viscoelastic layer to form a non-viscoelastic film. By contrast, an earlier example identified by Hook et al. demonstrated the structural transformation of a mussel adhesive protein adlayer, but this process was induced by chemical reaction with NaIO4 to cross-link a protein film . The case of the AH peptide has direct biological relevance because membrane association of the NS5A AH is necessary for HCV genome replication. Critically, this strategy also solves a major design challenge for fabricating planar bilayers on gold, which has a number of attractive electrical and optical properties.
In another study, the AH peptide-mediated process of vesicle rupture and planar bilayer formation was fit with a the Voigt-Voinova based model to analyze how the peptide interaction alters film properties including thickness, effective shear modulus, and shear viscosity . To fit the model, QCM-D resonance frequency and energy dissipation measurements of the AH peptide-mediated structural transformation were recorded at multiple overtones (Fig. 5g, h). Film thicknesses calculated by the Voigt-Voinova model confirmed that the Sauerbrey relationship—which describes the simple linear relationship between change in resonance frequency and mass adsorption in limited cases—is only valid for analyzing rigid films, and not soft layers (Fig. 5i). The results of this modeling confirm the importance of collecting a wide range of data. In this case, additional information enabled the change in mechanical properties of the thin film to be correlated with the AH peptide-membrane interaction. This combined approach may also be helpful for studying the mechanism of AH peptide’s vesicle-rupturing activity.
Based on the results of these studies, we observe several key findings. First, the maximum binding signal generated by adsorption of AH peptide to the intact vesicle platform is significantly greater than the signal on the planar bilayer platform. In part, this discrepancy may be due to differences in the structural features of a planar bilayer versus layer of intact vesicles. Indeed, in a fixed geometry, intact vesicles would have more available surface area for peptide binding than the planar bilayer. However, when the binding signals are normalized to account for the varying amount of lipid material in the different platforms, the binding signal on the intact vesicle platform is still 2.5 times greater. Depending on the target interaction, this platform design issue may be an important consideration for biosensing.
Second, the interaction kinetic are completely distinct in each case. On the planar bilayer platform, simple exponential adsorption kinetics is observed with minimal peptide binding. This behavior suggests that the interaction is nonspecifically mediated by the amphipathic nature of the peptide, and that membrane-associated AH peptide exists in the monomeric state. By contrast, interaction of AH peptide with intact vesicles promotes a structural transformation from intact vesicle layer to planar bilayer. The interaction kinetics are complex and show a two-step process. While the Voigt-Voinova model helped to determine the effects of the transformation process on film properties, additional evidence is necessary to understand the mechanism of this process. In this regard, recent work by Hatzakis et al.  on the intrinsic property of bilayers to recruit larger densities of amphipathic molecules when highly curved deserves attention. In addition, as the intact vesicle layer is the more biologically relevant model membrane system, the interaction kinetics observed in this system may be related to the role of NS5A protein in the HCV life cycle.
Third, the concentration-dependence of the AH peptide interaction varies between the platforms. At a concentration of 0.05 mg/mL, the AH peptide transforms a layer of intact vesicles into a planar bilayer. However, at the same concentration, the AH peptide does not bind to the planar bilayer. Higher concentrations of AH peptide are necessary to detect any binding signal on the planar bilayer platform. Taken together with the QCM-D responses, these data support that the transformation of an intact vesicle layer on titanium oxide or gold can result in the formation of a complete, planar bilayer with comparable sensing properties to those of a planar bilayer on the prototypical silicon oxide substrate. From an applications standpoint, the fabrication of a planar bilayer on these formerly intractable substrates is advantageous for the design of optical- and electrochemical-based sensors.
4.2 Multiple Technique Characterization of AH Peptide-Mediated Vesicle Rupture
While the assembly of a planar bilayer on gold and titanium oxide represents an important step towards the application of biological tools to solve problems in materials science, a more complete description of the fundamental mechanism behind the transformation process requires detailed study from multiple angles in order to evaluate its biomedical significance. Indeed, complex biological phenomena are difficult to interpret based on a single physical principle. In the case of the interaction between AH peptide and lipid vesicles, the QCM-D energy dissipation signal associated with the vesicle-to-bilayer transformation indicated an intermediate regime of high viscoelasticity. Such a change in film viscoelasticity might occur due to an increase in bound solvent resulting from vesicle swelling, for example. However, the QCM-D technique only senses acoustic mass, which is the combined mass of the adsorbed material (e.g., lipid and/or amino acid) as well as bound solvent. On the basis of this measurement alone, it is not possible to identify how the solvent mass fraction changes during the transformation. To address this challenge, it is therefore important to characterize biointerfacial processes by using several surface-sensitive techniques that measure different physical principles.
To investigate the mechanism by which AH peptide modulates the film properties of the intact vesicle layer, simultaneous QCM-D and reflectometry measurements to follow the AH peptide-mediated vesicle-to-bilayer transformation on gold have been reported . While the acoustic wave-based QCM-D technique characterizes the structural properties of an adlayer film, the optical-based reflectometry technique provides a measure of the bound molecular mass that is related to the effective thickness and the effective refractive index of the adlayer . When these two techniques are combined, several parameters can be simultaneously measured as a function of time, including: (1) adsorbed molecular mass (optical mass); (2) adsorbed molecular mass and solvent mass (acoustic mass); (3) effective film thickness; and (4) effective refractive index over the course of time . As a result, the solvent mass can be calculated by separating the acoustic and optical masses. Because vesicle swelling and expansion was initially hypothesized to be the cause of the AH peptide-mediated vesicle rupturing process, the ability to follow the change in solvent mass is particularly important to test this hypothesis and further understand the biological mechanism.
A magnified view of Fig. 6d is presented to highlight several key observations about the regimes of initial peptide interaction and vesicle rupturing (Fig. 6e). First, selection of the surface-sensitive technique is critical for detection of the target interaction. Only minor structural changes were detected by the QCM-D technique upon initial binding of AH peptide. By contrast, reflectometry was able to clearly identify and quantify this binding step. Second, saturation of AH peptide binding occurred before the swelling of vesicles reached saturation. This lag time of approximately 6 min suggests that membrane association of the AH peptide is insufficient to directly cause swelling and vesicle destabilization. Rather, some type of peptide rearrangement (e.g., pore formation) may occur to induce solvent uptake. Third, the optical mass measurement provides direct evidence that AH peptides bind to lipid vesicles.
Another benefit of the combined measurement approach is the ability to calculate a hydration signature to describe the role of solvent mass throughout the course of the interaction. To demonstrate how this parameter is useful for comparing macromolecular interactions, the self-assembly processes for planar bilayer formation on silicon oxide and gold were compared (Fig. 6f). An especially useful feature of hydration signature is the direct identification of individual kinetic steps to describe the overall mechanism of biological interactions [43, 95].
Taken together, the combination of QCM-D and reflectometry techniques enabled a more complete description of the AH peptide-mediated vesicle rupturing process. Large increases in adsorbed mass and energy dissipation are caused by swelling of the vesicles upon interaction with AH peptide. Based on the kinetic differences of the optical and acoustic mass responses, it appears that the binding of AH peptide is necessary but not sufficient to promote the structural transformation. Instead, the AH peptides presumably rearrange on the membrane interface to promote vesicle swelling and destabilization, leading to reassembly and planar bilayer formation. With these mechanistic details revealed, there is now a framework in place to evaluate the biomedical significance of this interaction and to engineer optimized peptides with more potent rupturing activity.
5 From Engineering to Biomedical Application
Based on the steps of platform design and fundamental characterization, the lipid vesicle-rupturing ability of the NS5A AH peptide was identified. Beyond the biological significance of this activity which remains to be determined, there are several important applications which directly emerge from this activity. First, there is now a strategy to form planar bilayers on previously intractable substrates that are important materials for biosensing. Second, AH peptide-mediated vesicle rupturing is a targetable activity of the NS5A protein that may serve as a surrogate for targeting its membrane association step and/or other biologically significant function. Third, the AH peptide demonstrates potential for rupturing lipid-enveloped virus particles that share similar membrane properties to lipid vesicles. In terms of direct impact, the virus particle-rupturing potential of the AH peptide is greatest because this inhibition strategy may serve as a broad-spectrum activity against all lipid-enveloped viruses or some subset thereof. Further, this rupturing activity would represent a completely novel mechanism of action that targets a conserved structural feature of the virus particle that is not encoded by the viral genome.
5.1 Vesicle Rupture as a Predictor of Virus Particle Lysis
To determine if the AH peptide possesses suitable properties for rupturing the lipid envelopes of virus particles, it is necessary to develop an appropriate model system to mimic the envelope structure. In this sense, intact lipid vesicles are an excellent choice because they replicate the membranous environment of lipid envelopes [50, 96–100]. Unlike the planar bilayer, intact vesicles also recapitulate membrane curvature. While the mechanism of the AH peptide’s vesicle rupturing activity was already determined by the combined QCM-D and reflectometry measurements, it was not determined how this in situ vesicle rupturing activity may correlate with in vitro virus particle rupturing.
Initial studies on the AH peptide-mediated rupturing process focused on vesicles with 50–60 nm diameters. This size range was selected because zwitterionic lipid vesicles of this diameter easily rupture on silicon oxide . For fundamental characterization of surface-specific adsorption kinetics, the vesicle size was therefore fixed and the variable parameter was the substrate material. This characterization work eventually led to the development of the planar bilayer and intact vesicle platforms which were used for probing AH peptide-mediated vesicle rupturing. In general, the different film properties of these two platforms are sufficient for studying how membrane properties affect these interactions. However, to further evaluate the AH peptide, the intact vesicle platform should be modified to better mimic the properties of lipid-enveloped virus particles. One of the key differences between the particles of different viruses is their size distribution, which is therefore a useful parameter to study. Indeed, one study recently used the intact vesicle platform with the QCM-D assay to determine the vesicle size range within which the AH peptide demonstrates rupturing activity in order to evaluate which viruses the AH peptide may target.
Taken together, these data demonstrate that the AH peptide possesses a selective targeting of small virus particles. Based on the results, a graph was constructed and identified that several Flaviviridae members including HCV are likely to be targeted by the AH peptide (Fig. 7e). Atomic force microscopy (AFM) measurements confirmed that AH peptide treatment can physically disrupt the lipid envelopes of viruses, including HCV, only within the target size range. While fusion peptides derived from viral envelope proteins have previously been shown to display antiviral activity by either destabilizing lipid envelopes or interfering with fusogenic domains of envelope proteins [102, 103], the AH peptide is neither involved in membrane fusion nor known to interact with other protein domains within the HCV NS5A protein. Furthermore, the NS5A protein is a nonstructural protein that is involved in intracellular HCV genome replication, and is not part of the HCV virion. Several antimicrobial peptides have also been described that physically disrupt viral envelopes .
By directly monitoring the physical effect of AH peptide treatment on enveloped viruses as well as confirming these findings with viral replication monitoring, the study demonstrated that the intact vesicle platform represents a reliable model system for predicting the susceptibility of individual viruses to AH peptide treatment. Predictions are most valid for viruses that more closely approximate key properties of synthetic lipid vesicles; including: (1) have a lipid envelope; and (2) minimal heterogeneity in particle size. Furthermore, it is not likely that the size corresponding to 100% vesicle rupture is the maximum size at which AH peptide can rupture virus particles. A more probable case is that the rupturing ability of the AH peptide against virus particles occurs on a continuum, as observed in Fig. 7c. Therefore, while the AH peptide may have a greater observed effect on smaller virus particles, it more likely still interacts with larger particles and may affect their infectivity even in the absence of direct rupturing. In addition to this method focused on determining the biophysical mechanism of the AH peptide-mediated vesicle rupturing process in order to predict therapeutic effect, other groups have followed up by confirming the in vitro activity of the AH peptide with molecular virology approaches.
5.2 Broad-Spectrum Virocidal Activity of AH Peptide
Another important feature identified by the authors was that C5A peptide can penetrate cell membranes (Fig. 8d) and has intracellular virocidal activity (Fig. 8e). Notably, there was no change in the amount of intracellular HCV RNA, confirming our finding that the AH peptide directly ruptures HCV particles. One point to note the report by Cheng et al. is that the peptide’s extracellular virocidal activity is more efficient than its intracellular activity. Bioavailability of peptide therapeutics is indeed a general challenge for effective drug concentrations to reach intracellular targets and may ultimately direct the peptide to extracellular applications such as prophylaxis. Nonetheless, in vivo studies in mice supported that the peptide has a promising toxicity profile and is non-immunogenic.
Virus susceptibility to AH peptide analogue treatment
HCV(JFH-I) genotype 2a
HCV (H77 envelope) genotype 1a
HCV (Con1 envelope) genotype 1b
HCV (J6CF envelope) genotype 2a
West Nile virus
Respiratory syncytial virus
Human immunodeficiency virus
Borna disease virus
Hepatitis B virus
Lymphocytic choriomeningitis virus
Vesicular stomatitis virus
Notably, some viruses including Influenza virus and Borna disease virus possess lipid envelopes and have particle size diameters near the 100% rupturing value, yet are not susceptible to treatment with AH peptide [99, 100]. More work is needed to fully elucidate the molecular determinants of virus susceptibility. Because many of the susceptible viruses such as HCV, West Nile virus, and dengue virus bud into the endoplasmic reticulum (ER) membrane, Cheng et al. attempted to explain their results by proposing, but not verifying, that the AH peptide targets membranes of certain lipid compositions. By contrast, our model provides conclusive evidence that the critical parameter for virus inhibition is the size of lipid-enveloped particles. Nonetheless, the composition of lipid envelopes may be a secondary factor and could be easily tested with the model membrane platform.
In summary, recent virological studies have confirmed that in situ characterization of AH peptide’s vesicle rupturing activity is a reliable predictor of in vitro virocidal activity against lipid-enveloped virus particles [44, 45]. In doing so, the key parameters of the AH peptide’s virocidal activity were found to be that: (1) amphipathic sequence is necessary; (2) lone cysteine residue is also necessary and may be involved in disulfide bond formation; and (3) variation in amino acid composition of the C5A peptides from the different HCV genotypes correlates with varying levels of virocidal activity. In terms of optimization, adjustment of the AH peptide sequence composition will therefore be useful to modulate antiviral potency and therapeutic index [46, 106, 107]. Indeed, Li et al.  recently described the rational design of a second-generation peptide that preserves potent anti-HCV and anti-HIV activities while minimizing hemolytic activity. Collectively, the results achieved thus far demonstrate the potential of engineering strategies for the characterization and targeting of membrane-associated steps in viral life cycles.
6 Outlook on Engineering Strategies for Antiviral Drug Development
The application of model membrane platforms to target the HCV NS5A protein has led to discovery of the broad-spectrum antiviral activity exhibited by AH peptide. As part of this process, fundamental characterization of the AH peptide-membrane interaction yielded a set of estimations for AH peptide’s virocidal activity, including the size range of lipid-enveloped virus particles which may be targeted. This activity was then validated by molecular virology studies and the AH peptide is now in the drug pipeline. In this outlook, we introduce areas where we envision engineering strategies may further aid antiviral drug development.
By assembling an in situ assay to predict virus particle rupture [40–43], the work reviewed here has established a cost-effective tool to rapidly screen and functionally characterize antiviral drug candidates in minutes rather than days. Based on this preliminary format, the system can be improved by increasing sensitivity to reduce the amount of peptide material required and/or adapting the signal response to a more readily high-throughput measurement technique.
As one alternative assay possibility, multiple studies have reported that the AH peptide can rupture the membranes of lipid vesicles, as observed by the release of encapsulated fluorescent dye [45, 108]. However, this assay would first need to be standardized before functional characterization and screening. Thus far, the reports describing this assay have used different lipid compositions, fluorescent dye reagents, and concentrations thereof. Further, the vesicle preparation methods have varied and there has been no physical characterization of vesicle properties. All of these parameters would affect the measurement response, and need to be controlled. These considerations raise awareness of the challenges for studying the biointerfacial sciences, and further demonstrate how engineering strategies such as those described here can streamline drug development and increase reproducibility and accuracy.
A significant benefit of the established QCM-D vesicle rupturing assay is its ability to directly characterize the physical properties of the intact vesicle platform. By this method, any major differences in the film property can be identified before experiment and minor differences can be normalized. Real-time monitoring of the interaction kinetics yields a distinct, complex signature based on a self-assembly process. As such, it is difficult to generate a false positive due to the complex signature, and yet it is also still practical to screen for this signature in a high-throughput format because it leads to a clear result, the self-assembly of a planar bilayer on gold with well-characterized mass and viscoelastic properties. At present, it is not known if there would be any correlation between the results of the QCM-D assay and a fluorescence-based membrane rupturing assay. Nonetheless, continued innovation in these types of functional assays will help to speed up drug discovery and development.
One area where these engineering assays would be useful is the characterization of other classes of antiviral drug candidates. Beyond lipid envelope-rupturing peptides, a small molecule compound has been reported to physically disrupt lipid membranes . Interestingly, this compound displays a general property of membrane disruption but only permanently damages the membranes of viruses and not host cells. Unlike the host cell which maintains a biogenic capacity to repair its lipid membranes , viruses do not have such reparative capacity [111–113]. As a result, this compound has a low toxicity profile and the chemical structure is amenable to optimization for improved pharmacokinetics. Along with the new class of phospholipid-mimicking RAFIs (Rigid Amphipathic Fusion Inhibitors)  and Arbidol—a broad-spectrum small molecule antiviral available in Russia and China [115–118]—the biophysical mechanism of these fusion inhibitors remains to be determined.
In terms of looking forward, we also believe that the NS5A-encoded AH represents a unique target. In addition to the zinc-binding domain of NS5A , the N-terminal AH is the sole, validated functional target. With the promising results of a recent Phase I clinical trial for the NS5A-targeting BMS-790052 compound against HCV infection in patients who followed a once-daily, dosing regimen , there is huge potential for developing additional inhibition strategies against the NS5A protein. Indeed, the amphipathic character of the AH is conserved across all HCV isolates to date [63, 64, 67], making the AH far less likely to evolve resistance mutations [120, 121]. The QCM-D vesicle rupturing assay could be alternatively used to screen chemical libraries for small molecules that inhibit vesicle rupturing by the NS5A AH peptide. On the same token, the broad-spectrum antiviral activity of the NS5A AH peptide itself is very attractive for many therapeutic uses, including as a broad-spectrum antiviral agent or even as an immune adjuvant .
In summary, engineering strategies offer a new approach to probe biological systems by designing physiologically relevant model platforms through bottom-up design. While the focus here has been on antiviral drug development, model membranes offer an excellent platform to study a wide range of membrane-associated biological processes. Since many of these activities are critical determinants of human health, it will be exciting to witness the application of these strategies to solve biomedical problems that are currently intractable.
The authors wish to acknowledge generous support from the National Research Foundation of Singapore, and Nanyang Technological University. This work was funded by the NRF Research Fellowship grant NRF–NRFF2011-01 (to NJC). JAJ is a recipient of the Nanyang President’s Graduate Scholarship.
- Popot JL, Engelman DM (2000) Annu Rev Biochem 69:881–922Google Scholar
- Simons K, Toomre D (2000) Natl Rev Mol Cell Biol 1(1):31–39Google Scholar
- Singer SJ, Nicolson GL (1972) Science 175(23):720–731Google Scholar
- Israelachvili JN, Marcelja S, Horn RG (1980) Q Rev Biophys 13(2):121–200Google Scholar
- Geiger B, Bershadsky A, Pankov R, Yamada KM (2001) Natl Rev Mol Cell Biol 2(11):793–805Google Scholar
- Smith A-S (2010) Nat Phys 6(10):726–729Google Scholar
- Hediger MA, Romero MF, Peng JB, Rolfs A, Takanaga H, Bruford EA (2004) Pflugers Arch 447(5):465–468Google Scholar
- Yildirim MA, Goh KI, Cusick ME, Barabasi AL, Vidal M (2007) Nat Biotechnol 25(10):1119–1126Google Scholar
- Haywood AM (1994) J Virol 68(1):1–5Google Scholar
- Miller S, Krijnse-Locker J (2008) Nat Rev Microbiol 6(5):363–374Google Scholar
- Salaita K, Nair PM, Petit RS, Neve RM, Das D, Gray JW, Groves JT (2010) Science 327(5971):1380–1385Google Scholar
- Eckert DM, Kim PS (2001) Annu Rev Biochem 70:777–810Google Scholar
- Jackman JA, Cho N-J, Duran RS, Frank CW (2009) Langmuir 26(6):4103–4112Google Scholar
- Garcia ML (2004) Nature 430(6996):153–155Google Scholar
- Mouritsen OG, Jörgensen K (1998) Pharm Res 15(10):1507–1519Google Scholar
- Turk B (2006) Nat Rev Drug Discov 5(9):785–799Google Scholar
- Arkin MR, Wells JA (2004) Nat Rev Drug Discov 3(4):301–317Google Scholar
- Tamm LK, Lai AL, Li Y (2007) Biochim Biophys Acta 1768(12):3052–3060Google Scholar
- Wang G (2008) Curr Protein Pept Sci 9(1):50–69Google Scholar
- Mariappan M, Mateja A, Dobosz M, Bove E, Hegde RS, Keenan RJ (2011) Nature 477(7362):61–66Google Scholar
- Tanford C, Reynolds JA (1976) Biochim Biophys Acta 457(2):133–170Google Scholar
- Ferrari E, Wright-Minogue J, Fang JW, Baroudy BM, Lau JY, Hong Z (1999) J Virol 73(2):1649–1654Google Scholar
- Yamashita T, Kaneko S, Shirota Y, Qin W, Nomura T, Kobayashi K, Murakami S (1998) J Biol Chem 273(25):15479–15486Google Scholar
- Hook F, Kasemo B, Grunze M, Zauscher S (2008) ACS Nano 2(12):2428–2436Google Scholar
- Grate JW, Frye GC (1996) Sens Update 2(1):37–83Google Scholar
- Cooper MA (2002) Nat Rev Drug Discov 1(7):515–528Google Scholar
- Moskovits M (1985) Rev Mod Phys 57(3):783Google Scholar
- Wang J (2005) Analyst 130(4):421–426Google Scholar
- Mrksich M (2008) ACS Nano 2(1):7–18Google Scholar
- Dufrene YF (2008) Nat Rev Micro 6(9):674–680Google Scholar
- Whitesides GM, Grzybowski B (2002) Science 295(5564):2418–2421Google Scholar
- Castner DG, Ratner BD (2002) Surf Sci 500(1–3):28–60Google Scholar
- Kasemo B (2002) Surf Sci 500(1–3):656–677Google Scholar
- Sackmann E (1996) Science 271(5245):43–48Google Scholar
- Chan Y-HM, Boxer SG (2007) Curr Opin Chem Biol 11(6):581–587Google Scholar
- Kiessling V, Domanska MK, Murray D, Wan C, Tamm LK (2008) In: Wiley encyclopedia of chemical biology, vol 4. Wiley, Hoboken, pp 411–422Google Scholar
- Nair PM, Salaita K, Petit RS, Groves JT (2011) Nat Protoc 6(4):523–539Google Scholar
- Bayley H, Cremer PS (2001) Nature 413(6852):226–230Google Scholar
- Castellana ET, Cremer PS (2006) Surf Sci Rep 61:429–444Google Scholar
- Cho NJ, Cho SJ, Cheong KH, Glenn JS, Frank CW (2007) J Am Chem Soc 129(33):10050–10051Google Scholar
- Cho NJ, Dvory-Sobol H, Xiong A, Cho SJ, Frank CW, Glenn JS (2009) ACS Chem Biol 4(12):1061–1067Google Scholar
- Cho NJ, Kanazawa KK, Glenn JS, Frank CW (2007) Anal Chem 79(18):7027–7035Google Scholar
- Cho NJ, Wang G, Edvardsson M, Glenn JS, Hook F, Frank CW (2009) Anal Chem 81(12):4752–4761Google Scholar
- Bobardt MD, Cheng G, de Witte L, Selvarajah S, Chatterji U, Sanders-Beer BE, Geijtenbeek TB, Chisari FV, Gallay PA (2008) Proc Natl Acad Sci USA 105(14):5525–5530Google Scholar
- Cheng G, Montero A, Gastaminza P, Whitten-Bauer C, Wieland SF, Isogawa M, Fredericksen B, Selvarajah S, Gallay PA, Ghadiri MR, Chisari FV (2008) Proc Natl Acad Sci USA 105(8):3088–3093Google Scholar
- de Witte L, Bobardt MD, Chatterji U, van Loenen FB, Verjans GM, Geijtenbeek TB, Gallay PA (2011) PLoS One 6(5):e18917Google Scholar
- Morens DM, Folkers GK, Fauci AS (2004) Nature 430(6996):242–249Google Scholar
- De Clercq E (2002) Nat Rev Drug Discov 1(1):13–25Google Scholar
- De Clercq E (2007) Nat Rev Drug Discov 6(12):1001–1018Google Scholar
- Penin F, Dubuisson J, Rey FA, Moradpour D, Pawlotsky JM (2004) Hepatology 39(1):5–19Google Scholar
- Murray CL, Jones CT, Rice CM (2008) Nat Rev Microbiol 6(9):699–708Google Scholar
- Lindenbach BD, Rice CM (2005) Nature 436(7053):933–938Google Scholar
- Moradpour D, Penin F, Rice CM (2007) Nat Rev Microbiol 5(6):453–463Google Scholar
- Froshauer S, Kartenbeck J, Helenius A (1988) J Cell Biol 107(6 Pt 1):2075–2086Google Scholar
- Egger D, Wolk B, Gosert R, Bianchi L, Blum HE, Moradpour D, Bienz K (2002) J Virol 76(12):5974–5984Google Scholar
- Suhy DA, Giddings TH Jr, Kirkegaard K (2000) J Virol 74(19):8953–8965Google Scholar
- Gosert R, Egger D, Lohmann V, Bartenschlager R, Blum HE, Bienz K, Moradpour D (2003) J Virol 77(9):5487–5492Google Scholar
- Gelman MA, Glenn JS (2010) Trends Mol Med 17(1):34–46Google Scholar
- Rong L, Dahari H, Ribeiro RM, Perelson AS (2010) Sci Transl Med 2(30):30ra32Google Scholar
- Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, Marumo F, Sato C (1995) J Clin Invest 96(1):224–230Google Scholar
- Macdonald A, Harris M (2004) J Gen Virol 85(Pt 9):2485–2502Google Scholar
- Sklan EH, Staschke K, Oakes TM, Elazar M, Winters M, Aroeti B, Danieli T, Glenn JS (2007) J Virol 81(20):11096–11105Google Scholar
- Elazar M, Cheong KH, Liu P, Greenberg HB, Rice CM, Glenn JS (2003) J Virol 77(10):6055–6061Google Scholar
- Brass V, Bieck E, Montserret R, Wolk B, Hellings JA, Blum HE, Penin F, Moradpour D (2002) J Biol Chem 277(10):8130–8139Google Scholar
- Schmitz U, Tan SL (2008) Recent Pat Antiinfect Drug Discov 3(2):77–92Google Scholar
- Tellinghuisen TL, Marcotrigiano J, Rice CM (2005) Nature 435(7040):374–379Google Scholar
- Penin F, Brass V, Appel N, Ramboarina S, Montserret R, Ficheux D, Blum HE, Bartenschlager R, Moradpour D (2004) J Biol Chem 279(39):40835–40843Google Scholar
- Tanaka M, Sackmann E (2005) Nature 437(7059):656–663Google Scholar
- Tamm LK, McConnell HM (1985) Biophys J 47(1):105–113Google Scholar
- Keller CA, Kasemo B (1998) Biophys J 75(3):1397–1402Google Scholar
- Zwang TJ, Fletcher WR, Lane TJ, Johal MS (2010) Langmuir 26(7):4598–4601Google Scholar
- Jung LS, Shumaker-Parry JS, Campbell CT, Yee SS, Gelb MH (2000) J Am Chem Soc 122(17):4177–4184Google Scholar
- Anderson TH, Min Y, Weirich KL, Zeng H, Fygenson D, Israelachvili JN (2009) Langmuir 25(12):6997–7005Google Scholar
- Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT (2004) Science 303(5657):495–499Google Scholar
- Reimhult E, Hook F, Kasemo B (2002) J Chem Phys 117:7401–7404Google Scholar
- Reimhult E, Hook F, Kasemo B (2002) Langmuir 19(5):1681–1691Google Scholar
- Mie Y, Suzuki M, Komatsu Y (2009) J Am Chem Soc 131(19):6646–6647Google Scholar
- Jadhav SR, Sui D, Garavito RM, Worden RM (2008) J Colloid Interface Sci 322(2):465–472Google Scholar
- Weiss SA, Bushby RJ, Evans SD, Henderson PJ, Jeuken LJ (2009) Biochem J 417(2):555–560Google Scholar
- Cho NJ, Cheong KH, Lee C, Frank CW, Glenn JS (2007) J Virol 81(12):6682–6689Google Scholar
- Rodahl M, Hook F, Fredriksson C, Keller CA, Krozer A, Brzezinski P, Voinova M, Kasemo B (1997) Faraday Discuss 107:229–246Google Scholar
- Cho NJ, Frank CW, Kasemo B, Hook F (2010) Nat Protoc 5(6):1096–1106Google Scholar
- Dixon MC (2008) J Biomol Tech 19(3):151–158Google Scholar
- Mechler A, Praporski S, Atmuri K, Boland M, Separovic F, Martin LL (2007) Biophys J 93(11):3907–3916Google Scholar
- Glasmastar K, Larsson C, Hook F, Kasemo B (2002) J Colloid Interface Sci 246(1):40–47Google Scholar
- Richter R, Mukhopadhyay A, Brisson A (2003) Biophys J 85(5):3035–3047Google Scholar
- Tyagi M, Rusnati M, Presta M, Giacca M (2001) J Biol Chem 276(5):3254–3261Google Scholar
- Mingeot-Leclercq MP, Deleu M, Brasseur R, Dufrene YF (2008) Nat Protoc 3(10):1654–1659Google Scholar
- Matsuzaki K, Murase O, Miyajima K (1995) Biochemistry 34(39):12553–12559Google Scholar
- Benachir T, Monette M, Grenier J, Lafleur M (1997) Eur Biophys J 25(3):201–210Google Scholar
- Hook F, Kasemo B, Nylander T, Fant C, Sott K, Elwing H (2001) Anal Chem 73(24):5796–5804Google Scholar
- Voinova MV et al (1999) Physica Scripta 59(5):391Google Scholar
- Hatzakis NS, Bhatia VK, Larsen J, Madsen KL, Bolinger PY, Kunding AH, Castillo J, Gether U, Hedegard P, Stamou D (2009) Nat Chem Biol 5(11):835–841Google Scholar
- Wang G, Rodahl M, Edvardsson M, Svedhem S, Ohlsson G, Hook F, Kasemo B (2008) Rev Sci Instrum 79(7):075107Google Scholar
- Edvardsson M, Svedhem S, Wang G, Richter R, Rodahl M, Kasemo B (2008) Anal Chem 81(1):349–361Google Scholar
- Aloia RC, Tian H, Jensen FC (1993) Proc Natl Acad Sci USA 90(11):5181–5185Google Scholar
- Baker KA, Dutch RE, Lamb RA, Jardetzky TS (1999) Mol Cell 3(3):309–319Google Scholar
- Calisher CH, Gould EA (2003) Adv Virus Res 59:1–19Google Scholar
- Harris A, Cardone G, Winkler DC, Heymann JB, Brecher M, White JM, Steven AC (2006) Proc Natl Acad Sci USA 103(50):19123–19127Google Scholar
- Kohno T, Goto T, Takasaki T, Morita C, Nakaya T, Ikuta K, Kurane I, Sano K, Nakai M (1999) J Virol 73(1):760–766Google Scholar
- Cho N-J, Jackman JA, Liu M, Frank CW (2011) Langmuir 27(7):3739–3748Google Scholar
- Joanne P, Nicolas P, El Amri C (2009) Protein Pept Lett 16(7):743–750Google Scholar
- Shai Y (2000) Biosci Rep 20(6):535–555Google Scholar
- Jenssen H, Hamill P, Hancock RE (2006) Clin Microbiol Rev 19(3):491–511Google Scholar
- Jaeckel E, Cornberg M, Wedemeyer H, Santantonio T, Mayer J, Zankel M, Pastore G, Dietrich M, Trautwein C, Manns MP (2001) N Engl J Med 345(20):1452–1457Google Scholar
- Li GR, He LY, Liu XY, Liu AP, Huang YB, Qiu C, Zhang XY, Xu JQ, Yang W, Chen YX (2011) Chem Biol Drug Des 78(5):835–843Google Scholar
- Lin Q, Fang D, Hou X, Le Y, Fang J, Wen F, Gong W, Chen K, Wang JM, Su SB (2011) J Immunol 186(4):2087–2094Google Scholar
- Palomares-Jerez MF, Guillen J, Villalain J (2010) Biochim Biophys Acta 1798(6):1212–1224Google Scholar
- Wolf MC, Freiberg AN, Zhang T, Akyol-Ataman Z, Grock A, Hong PW, Li J, Watson NF, Fang AQ, Aguilar HC, Porotto M, Honko AN, Damoiseaux R, Miller JP, Woodson SE, Chantasirivisal S, Fontanes V, Negrete OA, Krogstad P, Dasgupta A, Moscona A, Hensley LE, Whelan SP, Faull KF, Holbrook MR, Jung ME, Lee B (2010) Proc Natl Acad Sci USA 107(7):3157–3162Google Scholar
- Holthuis JC, Levine TP (2005) Natl Rev Mol Cell Biol 6(3):209–220Google Scholar
- McNeil PL, Steinhardt RA (2003) Annu Rev Cell Dev Biol 19:697–731Google Scholar
- McNeil PL, Terasaki M (2001) Nat Cell Biol 3(5):E124–E129Google Scholar
- Meldolesi J (2003) J Cell Mol Med 7(3):197–203Google Scholar
- St Vincent MR, Colpitts CC, Ustinov AV, Muqadas M, Joyce MA, Barsby NL, Epand RF, Epand RM, Khramyshev SA, Valueva OA, Korshun VA, Tyrrell DL, Schang LM (2010) Proc Natl Acad Sci USA 107(40):17339–17344Google Scholar
- Boriskin YS, Pecheur EI, Polyak SJ (2006) Virol J 3:56Google Scholar
- Shi L, Xiong H, He J, Deng H, Li Q, Zhong Q, Hou W, Cheng L, Xiao H, Yang Z (2007) Arch Virol 152(8):1447–1455Google Scholar
- Teissier E, Zandomeneghi G, Loquet A, Lavillette D, Lavergne JP, Montserret R, Cosset FL, Bockmann A, Meier BH, Penin F, Pecheur EI (2011) PLoS One 6(1):e15874Google Scholar
- Villalain J (2010) J Phys Chem B 114(25):8544–8554Google Scholar
- Gao M, Nettles RE, Belema M, Snyder LB, Nguyen VN, Fridell RA, Serrano-Wu MH, Langley DR, Sun JH, O’Boyle DR 2nd, Lemm JA, Wang C, Knipe JO, Chien C, Colonno RJ, Grasela DM, Meanwell NA, Hamann LG (2010) Nature 465(7294):96–100Google Scholar
- Fridell RA, Qiu D, Valera L, Wang C, Rose RE, Gao M (2011) J Virol 85(14):7312–7320Google Scholar
- Fridell RA, Qiu D, Wang C, Valera L, Gao M (2010) Antimicrob Agents Chemother 54(9):3641–3650Google Scholar
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