Peptide-Based Flavivirus Biosensors: From Cell Structure to Virological and Serological Detection Methods.

In tropical and developing countries, mosquito-borne diseases by flaviviruses pose a serious threat to public health. Early detection is critical for preventing their spread, but conventional methods are time-consuming and require skilled technicians. Biosensors have been developed to address this issue, but cross-reactivity with other flaviviruses remains a challenge. Peptides are essentially biomaterials used in diagnostics that allow virological and serological techniques to identify flavivirus selectively. This biomaterial originated as a small protein consisting of two to 50 amino acid chains. They offer flexibility in chemical modification and can be easily synthesized and applied to living cells in the engineering process. Peptides could potentially be developed as robust, low-cost, sensitive, and selective receptors for detecting flaviviruses. However, modification and selection of the receptor agents are crucial to determine the effectiveness of binding between the targets and the receptors. This paper addresses two potential peptide nucleic acids (PNAs) and affinity peptides that can detect flavivirus from another target-based biosensor as well as the potential peptide behaviors of flaviviruses. The PNAs detect flaviviruses based on the nucleotide base sequence of the target's virological profile via Watson-Crick base pairing, while the affinity peptides sense the epitope or immunological profile of the targets. Recent developments in the functionalization of peptides for flavivirus biosensors are explored in this Review by division into electrochemical, optical, and other detection methods.

Phospholipid binding of the dengue virus envelope E protein segment containing the conserved His residue.

Flaviviruses encompass many important human pathogens, including Dengue, Zika, West Nile, Yellow fever, Japanese encephalitis, and Tick-borne encephalitis viruses as well as several emerging viruses that affect millions of people worldwide. They enter cells by endocytosis, fusing their membrane with the late endosomal one in a pH-dependent manner, so membrane fusion is one of the main targets for obtaining new antiviral inhibitors. The envelope E protein, a class II membrane fusion protein, is responsible for fusion and contains different domains involved in the fusion mechanism, including the fusion peptide. However, other segments, apart from the fusion peptide, have been implicated in the mechanism of membrane fusion, in particular a segment containing a His residue supposed to act as a specific pH sensor. We have used atomistic molecular dynamics to study the binding of the envelope E protein segment containing the conserved His residue in its three different tautomer forms with a complex membrane mimicking the late-endosomal one. We show that this His-containing segment is capable of spontaneous membrane binding, preferentially binds electronegatively charged phospholipids and does not bind cholesterol. Since Flaviviruses have caused epidemics in the past, continue to do so and will undoubtedly continue to do so, this specific segment could characterise a new target that would allow finding effective antiviral molecules against DENV virus in particular and Flaviviruses in general.

An Absolutely Conserved Tryptophan in the Stem of the Envelope Protein E of Flaviviruses Is Essential for the Formation of Stable Particles.

The major envelope protein E of flaviviruses contains an ectodomain that is connected to the transmembrane domain by the so-called "stem" region. In mature flavivirus particles, the stem is composed of two or three mostly amphipathic α-helices and a conserved sequence element (CS) with an undefined role in the viral life cycle. A tryptophan is the only residue within this region which is not only conserved in all vector-borne flaviviruses, but also in the group with no known vector. We investigated the importance of this residue in different stages of the viral life cycle by a mutagenesis-based approach using tick-borne encephalitis virus (TBEV). Replacing W421 by alanine or histidine strongly reduced the release of infectious virions and their thermostability, whereas fusion-related entry functions and virus maturation were still intact. Serial passaging of the mutants led to the emergence of a same-site compensatory mutation to leucine that largely restored these properties of the wildtype. The conserved tryptophan in CS (or another big hydrophobic amino acid at the same position) is thus essential for the assembly and infectivity of flaviviruses by being part of a network required for conferring stability to infectious particles.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry.

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Broad-Spectrum Flavivirus Inhibitors: a Medicinal Chemistry Point of View.

Infections by flaviviruses, such as Dengue, West Nile, Yellow Fever and Zika viruses, represent a growing risk for global health. There are vaccines only for few flaviviruses while no effective treatments are available. Flaviviruses share epidemiological, structural, and ecologic features and often different viruses can co-infect the same host. Therefore, the identification of broad-spectrum inhibitors is highly desirable either for known flaviviruses or for viruses that likely will emerge in the future. Strategies targeting both virus and host factors have been pursued to identify broad-spectrum antiflaviviral agents. In this review, we describe the most promising and best characterized targets and their relative broad-spectrum inhibitors, identified by drug repurposing/libraries screenings and by focused medicinal chemistry campaigns. Finally, we discuss about future strategies to identify new broad-spectrum antiflavivirus agents.

Development and characterization of specific anti-Usutu virus chicken-derived single chain variable fragment antibodies.

Usutu virus belongs to the Japanese encephalitis serogroup within the Flaviviridae family. Mammals may become incidental hosts after the bite of an infected mosquito while birds act as the main reservoir. Human cases have become more common recently and elicit various outcomes ranging from asymptomatic to severe illness including encephalitis. Problematically, antisera against Usutu virus cross-react with other flaviviruses such as the co-circulating West Nile virus. As an approach to generate Usutu virus-specific antibodies, we immunized chickens with purified Usutu virus envelope protein domain III, isolated the spleen mRNA and generated an scFv phage display library. The most potent binders for Usutu virus domain III were selected via biopanning and their affinity to domain III was examined using SPR. Four scFvs bound the domain III of Usutu virus in the nanomolar region; two bound the protein over 40 times more strongly than West Nile virus domain III. We further characterized these scFv antibodies for suitability in standard laboratory tests such as western blots, ELISA, and neutralization tests. Four specific and one cross-reactive antibody performed well in western blots with domain III and the full-length envelope protein of Usutu virus and West Nile virus. All antibodies bound in virus ELISA assays to Usutu virus strain Vienna-2001. However, none of the antibodies neutralized either Usutu virus or West Nile virus. These antibody candidates could be crucial in future diagnostic tests to distinguish Usutu virus from other flaviviruses and might even offer virus neutralization after a conversion to Fab or IgG.

Identification of a Neutralizing Monoclonal Antibody That Recognizes a Unique Epitope on Domain III of the Envelope Protein of Tembusu Virus.

Domain III of the envelope protein (EDIII) is the major target of flavivirus neutralizing antibody. To date, little is known regarding antibody-mediated neutralization of Tembusu virus (TMUV), a novel flavivirus emerging in duck in 2010. Here, a novel monoclonal antibody (MAb), designated 12F11, was prepared by immunization of mice with recombinant EDIII (rEDIII) protein. Using virus neutralization test, 12F11 in undiluted ascites neutralized the TMUV infectivity to induce the development of cytopathic effects in BHK-21 cells, indicating that 12F11 exhibits a neutralizing activity. The neutralizing activity of 12F11 was confirmed by plaque reduction neutralization test, in which 12F11 reduced significantly the number of plaques produced by TMUV in BHK-21 cells. Western blot analyses of a series of truncated rEDIII proteins showed that the epitope recognized by 12F11 includes amino acids between residues 8 and 77 of EDIII protein. Function analysis demonstrated that 12F11 neutralizes TMUV infection at virus adsorption and at a step after adsorption to a certain extent. The present study provides an important step towards elucidating antibody-mediated neutralization of TMUV.

The truncated E protein of DTMUV provide protection in young ducks.

Duck Tembusu virus (DTMUV) is a major pathogen of duck industry in China. In the current study, we generated different constructs containing envelope (E) protein, pre-membrane-envelope (prM-E) protein, and C-terminally truncated E protein of the DTMUV. The constructed proteins could induce specific antibody responses in young ducks. When ducklings were immunized with the constructed proteins, they were 100% protected against DTMUV infection. Furthermore, the fluorescent signal of the truncated E protein was stronger than other constructed proteins, when Bac-to-Bac baculovirus expression system was applied. Our data demonstrated that the truncated E protein used in the current study could be applied as a potential vaccine candidate to control DTMUV infection in young ducks.

Molecular Basis of a Protective/Neutralizing Monoclonal Antibody Targeting Envelope Proteins of both Tick-Borne Encephalitis Virus and Louping Ill Virus.

Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are members of the tick-borne flaviviruses (TBFVs) in the family Flaviviridae which cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines against TBEV and LIV are available, infection rates are rising due to the low vaccination coverage. To date, no specific therapeutics have been licensed. Several neutralizing monoclonal antibodies (MAbs) show promising effectiveness in the control of TBFVs, but the underlying molecular mechanisms are yet to be characterized. Here, we determined the crystal structures of the LIV envelope (E) protein and report the comparative structural analysis of a TBFV broadly neutralizing murine MAb (MAb 4.2) in complex with either the LIV or TBEV E protein. The structures reveal that MAb 4.2 binds to the lateral ridge of domain III of the E protein (EDIII) of LIV or TBEV, an epitope also reported for other potently neutralizing MAbs against mosquito-borne flaviviruses (MBFVs), but adopts a unique binding orientation. Further structural analysis suggested that MAb 4.2 may neutralize flavivirus infection by preventing the structural rearrangement required for membrane fusion during virus entry. These findings extend our understanding of the vulnerability of TBFVs and other flaviviruses (including MBFVs) and provide an avenue for antibody-based TBFV antiviral development.IMPORTANCE Understanding the mechanism of antibody neutralization/protection against a virus is crucial for antiviral countermeasure development. Tick-borne encephalitis virus (TBEV) and louping ill virus (LIV) are tick-borne flaviviruses (TBFVs) in the family Flaviviridae They cause encephalomeningitis and encephalitis in humans and other animals. Although vaccines for both viruses are available, infection rates are rising due to low vaccination coverage. In this study, we solved the crystal structures of the LIV envelope protein (E) and a broadly neutralizing/protective TBFV MAb, MAb 4.2, in complex with E from either TBEV or LIV. Key structural features shared by TBFV E proteins were analyzed. The structures of E-antibody complexes showed that MAb 4.2 targets the lateral ridge of both the TBEV and LIV E proteins, a vulnerable site in flaviviruses for other potent neutralizing MAbs. Thus, this site represents a promising target for TBFV antiviral development. Further, these structures provide important information for understanding TBFV antigenicity.

Crystal structure of Usutu virus envelope protein in the pre-fusion state.

BACKGROUND: Usutu virus (USUV) is a mosquito-born flavivirus that can infect multiple avian and mammalian species. The viral surface envelope (E) protein functions to initiate the viral infection by recognizing cellular receptors and mediating the subsequent membrane fusion, and is therefore a key virulence factor involved in the pathogenesis of USUV. The structural features of USUV-E, however, remains un-investigated thus far. FINDINGS: Using the crystallographic method, we determined the structure of USUV-E in the pre-fusion state at 2.0 angstrom. As expected, the overall fold of USUV-E, with three ß-barrel domains (DI, DII, and DIII), resembles those of other flaviviral E proteins. In comparison to other pre-fusion E structures, however, USUV-E exhibits an apparently enlarged inter-domain angle between DI and DII, leading to a more extended conformation. Using our structure and other reported pre-fusion E structures, the DI-DII domain-angle difference was analyzed in a pairwise manner. The result shows a much higher degree of variations for USUV-E, indicating the potential for remarkable DI-DII domain angle plasticity among flaviviruses. CONCLUSION: We report the crystal structure of USUV-E and show that its pre-fusion structure has an enlarged DI-DII domain-angle which has not been observed in other reported flaviviral E-structures.

Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein.

Viral envelope proteins are required for productive viral entry and initiation of infection. Although the humoral immune system provides ample evidence for targeting envelope proteins as an antiviral strategy, there are few pharmacological interventions that have this mode of action. In contrast to classical antiviral targets such as viral proteases and polymerases, viral envelope proteins as a class do not have a well-conserved active site that can be rationally targeted with small molecules. We previously identified compounds that inhibit dengue virus by binding to its envelope protein, E. Here, we show that these small molecules inhibit dengue virus fusion and map the binding site of these compounds to a specific pocket on E. We further demonstrate inhibition of Zika, West Nile, and Japanese encephalitis viruses by these compounds, providing pharmacological evidence for the pocket as a target for developing broad-spectrum antivirals against multiple, mosquito-borne flavivirus pathogens.

A Funneled Conformational Landscape Governs Flavivirus Fusion Peptide Interaction with Lipid Membranes.

During host cell infection by flaviviruses such as dengue and Zika, acidic pH within the endosome triggers a conformational change in the envelope protein on the outer surface of the virion. This results in exposure of the ∼15 residue fusion peptide (FP) region, freeing it to induce fusion between the viral and endosomal membranes. A better understanding of the conformational dynamics of the FP in the presence of membranes, and the basis for its selectivity for anionic lipid species present within the endosome, would facilitate its therapeutic targeting with antiviral drugs and antibodies. In this work, multiscale modeling, simulations, and free energy calculations (including a total of ∼75 µs of atomic-resolution sampling), combined with imaging total internal reflection fluorescence correlation spectroscopy experiments, were employed to investigate the mechanisms of interaction of FP variants with lipid bilayers. Wild-type FPs (in the presence or absence of a fluorescein isothiocyanate tag) were shown to possess a funneled conformational landscape governing their exit from solvent and penetration into the lipid phase and to exhibit an electrostatically favored >2-fold affinity for membranes containing anionic species over purely zwitterionic ones. Conversely, the landscape was abolished in a nonfunctional point mutant, leading to a 2-fold drop in host membrane affinity. Collectively, our data reveal how the highly conserved flavivirus FP has evolved to funnel its conformational space toward a maximally fusogenic state anchored within the endosomal membrane. Therapeutically targeting the accessible ensemble of FP conformations may represent a new, rational strategy for blocking viral infection.

Structures and Functions of the Envelope Glycoprotein in Flavivirus Infections.

Flaviviruses are enveloped, single-stranded RNA viruses that widely infect many animal species. The envelope protein, a structural protein of flavivirus, plays an important role in host cell viral infections. It is composed of three separate structural envelope domains I, II, and III (EDI, EDII, and EDIII). EDI is a structurally central domain of the envelope protein which stabilizes the overall orientation of the protein, and the glycosylation sites in EDI are related to virus production, pH sensitivity, and neuroinvasiveness. EDII plays an important role in membrane fusion because of the immunodominance of the fusion loop epitope and the envelope dimer epitope. Additionally, EDIII is the major target of neutralization antibodies. The envelope protein is an important target for research to develop vaccine candidates and antiviral therapeutics. This review summarizes the structures and functions of ED I/II/III, and provides practical applications for the three domains, with the ultimate goal of implementing strategies to utilize the envelope protein against flavivirus infections, thus achieving better diagnostics and developing potential flavivirus therapeutics and vaccines.

Engineering of Chimeric Protein Based on E Protein Domain III of Tick- Borne Encephalitis Virus and OmpF Porin of Yersinia pseudotuberculosis.

BACKGROUND: Tick-borne encephalitis poses a serious public health threat in the endemic regions. The disease treatment is restricted to symptomatic therapy, so great expectations are in the development of the prophylactic and therapeutic vaccines. The domain III of E protein of the tickborne encephalitis virus is the main antigenic domain which includes virus-specific epitopes recognized by neutralizing antibodies. OBJECTIVES: The main objective of this study was to design, express, isolate and characterize the chimeric protein based on the fusion of domain III of E protein of the tick-borne encephalitis virus and bacterial porin OmpF from Yersinia pseudotuberculosis. METHODS: The chimeric gene was obtained by the PCR based fusion method from two fragments containing overlapping linker sequences. Resulting plasmids were transformed into BL21(DE3) pLysS electrocompetent cells for subsequent heterologous protein expression. All recombinant proteins were purified using immobilized metal affinity chromatography under denaturing conditions. The identity of the chimeric protein was confirmed by MALDI-TOF mass spectrometry and immunoblot analysis. The content of antibodies against the EIII protein was estimated in mice blood serum by ELISA. RESULTS: The bacterial partner protein was used for decreasing toxicity and increasing immunogenicity of antigen. The chimeric protein was successfully expressed by the Escherichia coli cells. The purified protein was recognized with immunoblots by anti-E protein of tick-borne encephalitis virus monoclonal antibodies. Furthermore, the protein was able to elicit antibody response against domain III of E protein in immunized mice. CONCLUSION: The newly obtained chimeric antigen could be valuable for the development of the preventing tick-borne encephalitis subunit vaccines.

Flavivirus structural heterogeneity: implications for cell entry.

The explosive spread of Zika virus is the most recent example of the threat imposed to human health by flaviviruses. High-resolution structures are available for several of these arthropod-borne viruses, revealing alternative icosahedral organizations of immature and mature virions. Incomplete proteolytic maturation, however, results in a cloud of highly heterogeneous mosaic particles. This heterogeneity is further expanded by a dynamic behavior of the viral envelope glycoproteins. The ensemble of heterogeneous and dynamic infectious particles circulating in infected hosts offers a range of alternative possible receptor interaction sites at their surfaces, potentially contributing to the broad flavivirus host-range and variation in tissue tropism. The potential synergy between heterogeneous particles in the circulating cloud thus provides an additional dimension to understand the unanticipated properties of Zika virus in its recent outbreaks.

Characterization of the Zika virus two-component NS2B-NS3 protease and structure-assisted identification of allosteric small-molecule antagonists.

The recent re-emergence of Zika virus (ZIKV)1, a member of the Flaviviridae family, has become a global emergency. Currently, there are no effective methods of preventing or treating ZIKV infection, which causes severe neuroimmunopathology and is particularly harmful to the developing fetuses of infected pregnant women. However, the pathology induced by ZIKV is unique among flaviviruses, and knowledge of the biology of other family members cannot easily be extrapolated to ZIKV. Thus, structure-function studies of ZIKV proteins are urgently needed to facilitate the development of effective preventative and therapeutic agents. Like other flaviviruses, ZIKV expresses an NS2B-NS3 protease, which consists of the NS2B cofactor and the NS3 protease domain and is essential for cleavage of the ZIKV polyprotein precursor and generation of fully functional viral proteins. Here, we report the enzymatic characterization of ZIKV protease, and we identify structural scaffolds for allosteric small-molecule inhibitors of this protease. Molecular modeling of the protease-inhibitor complexes suggests that these compounds bind to the druggable cavity in the NS2B-NS3 protease interface and affect productive interactions of the protease domain with its cofactor. The most potent compound demonstrated efficient inhibition of ZIKV propagation in vitro in human fetal neural progenitor cells and in vivo in SJL mice. The inhibitory scaffolds could be further developed into valuable research reagents and, ultimately, provide a roadmap for the selection of efficient inhibitors of ZIKV infection.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 µM, 11.4 µM, and 4.8 µM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 µM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

Oligomeric viral proteins: small in size, large in presence.

Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.

Structures of the Zika Virus Envelope Protein and Its Complex with a Flavivirus Broadly Protective Antibody.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a current global public health concern. The flavivirus envelope (E) glycoprotein is responsible for virus entry and represents a major target of neutralizing antibodies for other flaviviruses. Here, we report the structures of ZIKV E protein at 2.0 Å and in complex with a flavivirus broadly neutralizing murine antibody 2A10G6 at 3.0 Å. ZIKV-E resembles all the known flavivirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity in vitro and neutralizes currently circulating ZIKV strains in vitro and in mice. The E protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV.