The step-by-step assembly mechanism of secreted flavivirus NS1 tetramer and hexamer captured at atomic resolution.

Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic ß-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.

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.

A Non-Active-Site Inhibitor with Selectivity for Zika Virus NS2B-NS3 Protease.

Flavivirus infection usually results in fever accompanied by headache, arthralgia, and, in some cases, rash. Although the symptoms are mild, full recovery can take several months. Flaviviruses encode seven nonstructural proteins that represent potential drug targets for this viral family. Focusing on the Zika virus NS2B-NS3 protease, we uncovered a unique inhibitor, MH1, composed of aminothiazolopyridine and benzofuran moieties. MH1 inhibits ZVP with a biochemical IC50 of 440 nM and effectively blocks cleavage of ZVP substrates in cells. Surprisingly, MH1 inhibits the other flaviviral proteases at least 18-fold more weakly. This same phenomenon was observed in assays of the viral cytopathic effect, where only Zika virus showed sensitivity to MH1. This selectivity was unexpected since flaviviral proteases have high similarity in sequence and protein structure. MH1 binds at an allosteric site, as demonstrated by its ability to stabilize ZVP synergistically with an active site inhibitor. To understand its selectivity, we constructed a series of hybrid proteases composed of select segments of ZVP, which is sensitive to MH1, and dengue virus protease, which is essentially insensitive to MH1. Our results suggest that MH1 binds to the NS3 protease domain, disrupting its interaction with NS2B. These interactions are essential for substrate binding and cleavage. In particular, the unique dynamic properties of NS2B from Zika seem to be required for the function of MH1. Insights into the mechanism of MH1 function will aid us in developing non-active-site-directed, pan-flaviviral inhibitors, by highlighting the importance of evaluating and considering the dynamics of the NS2B regions.

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.

Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease.

Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.

Hidden symmetry of the flavivirus protein shell and pH-controlled reconstruction of the viral surface.

Using recent Zika virus structural data we reveal a hidden symmetry of protein order in immature and mature flavivirus shells, violating the Caspar-Klug paradigmatic model of capsid structures. We show that proteins of the outer immature shell layer exhibit trihexagonal tiling, while proteins from inner and outer layers conjointly form a double-shelled close-packed structure, based on a common triangular spherical lattice. Within the proposed structural model, we furthermore rationalize the structural organization of misassembled non-infectious subviral particles that have no inner capsid. We consider a pH-controlled structural reconstruction of the outer shell from the trimeric to the dimeric state, and demonstrate that this transition, occurring during the virus maturation, can be induced by changes in protein charges at lower pH, leading to a decrease in the electrostatic interaction free energy. This transition could also be assisted by electrostatic attraction of shell proteins to the interposed lipid membrane substrate separating the shells.

Dengue Virus Entry/Fusion Inhibition By Small Bioactive Molecules: A Critical Review.

Many flaviviruses are remarkable human pathogens that can be transmitted by mosquitoes and ticks. Despite the availability of vaccines for viral infections such as yellow fever, Japanese encephalitis, and tick-borne encephalitis, flavivirus-like dengue is still a significant life-threatening illness worldwide. To date, there is no antiviral treatment for dengue therapy. Industry and the research community have been taking ongoing steps to improve anti-flavivirus treatment to meet this clinical need. The successful activity has been involved in the inhibition of the virus entry fusion process in the last two decades. In this study, the latest understanding of the use of small molecules used as fusion inhibitors has been comprehensively presented. We summarized the structure, the process of fusion of dengue virus E protein (DENV E), and the amino acids involved in the fusion process. Special attention has been given to small molecules that allow conformational changes to DENV E protein, viz. blocking the pocket of ßOG, which is important for fusion.

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.

Chimeric Flaviviral RNA-siRNA Molecules Resist Degradation by The Exoribonuclease Xrn1 and Trigger Gene Silencing in Mammalian Cells.

RNA is an emerging platform for drug delivery, but the susceptibility of RNA to nuclease degradation remains a major barrier to its implementation in vivo. Here, we engineered flaviviral Xrn1-resistant RNA (xrRNA) motifs to host small interfering RNA (siRNA) duplexes. The xrRNA-siRNA molecules self-assemble in vitro, resist degradation by the conserved eukaryotic 5' to 3' exoribonuclease Xrn1, and trigger gene silencing in 293T cells. The resistance of the molecules to Xrn1 does not translate to stability in blood serum. Nevertheless, our results demonstrate that flavivirus-derived xrRNA motifs can confer Xrn1 resistance on a model therapeutic payload and set the stage for further investigations into using the motifs as building blocks in RNA nanotechnology.

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.

Roles of the Endogenous Lunapark Protein during Flavivirus Replication.

The endoplasmic reticulum (ER) of eukaryotic cells is a dynamic organelle, which undergoes continuous remodeling. At the three-way tubular junctions of the ER, the lunapark (LNP) protein acts as a membrane remodeling factor to stabilize these highly curved membrane junctions. In addition, during flavivirus infection, the ER membrane is invaginated to form vesicles (Ve) for virus replication. Thus, LNP may have roles in the generation or maintenance of the Ve during flavivirus infection. In this study, our aim was to characterize the functions of LNP during flavivirus infection and investigate the underlying mechanisms of these functions. To specifically study virus replication, we generated cell lines expressing replicons of West Nile virus (Kunjin strain) or Langat virus. By using these replicon platforms and electron microscopy, we showed that depletion of LNP resulted in reduced virus replication, which is due to its role in the generation of the Ve. By using biochemical assays and high-resolution microscopy, we found that LNP is recruited to the Ve and the protein interacts with the nonstructural protein (NS) 4B. Therefore, these data shed new light on the interactions between flavivirus and host factors during viral replication.

Computational modelling of flavivirus dynamics: The ins and outs.

Enveloped viruses such as the flaviviruses represent a significant burden to human health around the world, with hundreds of millions of people each year affected by dengue alone. In an effort to improve our understanding of the molecular basis for the infective mechanisms of these viruses, extensive computational modelling approaches have been applied to elucidate their conformational dynamics. Multiscale protocols have been developed to simulate flavivirus envelopes in close accordance with biophysical data, in particular derived from cryo-electron microscopy, enabling high-resolution refinement of their structures and elucidation of the conformational changes associated with adaptation both to host environments and to immunological factors such as antibodies. Likewise, integrative modelling efforts combining data from biophysical experiments and from genome sequencing with chemical modification are providing unparalleled insights into the architecture of the previously unresolved nucleocapsid complex. Collectively, this work provides the basis for the future rational design of new antiviral therapeutics and vaccine development strategies targeting enveloped viruses.

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.

A Simple Method for the Design and Development of Flavivirus NS1 Recombinant Proteins Using an In Silico Approach.

Even in countries that are currently not facing a flavivirus epidemic, the spread of mosquito-borne flaviviruses presents an increasing public threat, owing to climate change, international travel, and other factors. Many of these countries lack the resources (viral strains, clinical specimens, etc.) needed for the research that could help cope with the threat imposed by flaviviruses, and therefore, an alternative approach is needed. Using an in silico approach to global databases, we aimed to design and develop flavivirus NS1 recombinant proteins with due consideration towards antigenic variation. NS1 genes analyzed in this study included a total of 6,823 sequences, from Dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus (WNV), Zika virus (ZIKV), and Yellow fever virus (YKV). We extracted and analyzed 316 DENV NS1 sequence types (STs), 59 JEV STs, 75 WNV STs, 30 YFV STs, and 43 ZIKV STs using a simple algorithm based on phylogenetic analysis. STs were reclassified according to the variation of the major epitope by MHC II binding. 78 DENV epitope type (EpT), 29 JEV EpTs, 29 WNV EpTs, 12 YFV EpTs, and 5 ZIKV EpTs were extracted according to their major epitopes. Also, frequency results showed that there were dominant EpTs in all flavivirus. Fifteen STs were selected and purified for the expression of recombinant antigen in Escherichia coli by sodium dodecyl sulfate extraction. Our study details a novel in silico approach for the development of flavivirus diagnostics, including a simple way to screen the important peptide regions.

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.

Structure-guided paradigm shifts in flavivirus assembly and maturation mechanisms.

The flavivirus genus encompasses more than 75 unique viruses, including dengue virus which accounts for almost 390 million global infections annually. Flavivirus infection can result in a myriad of symptoms ranging from mild rash and flu-like symptoms, to severe encephalitis and even hemorrhagic fever. Efforts to combat the impact of these viruses have been hindered due to limited antiviral drug and vaccine development. However, the advancement of knowledge in the structural biology of flaviviruses over the last 25 years has produced unique perspectives for the identification of potential therapeutic targets. With particular emphasis on the assembly and maturation stages of the flavivirus life cycle, it is the goal of this review to comparatively analyze the structural similarities between flaviviruses to provide avenues for new research and innovation.

Systematic Analysis of Structure Similarity between Zika Virus and Other Flaviviruses.

Zika virus (ZIKV) infection has caused global concern because of its association with fetal microcephaly and serious neurological complications in adults since 2016. Currently, no specific anti-ZIKV therapy is available to control ZIKV infection. During the last couple of years, the intensive investigation of ZIKV structure has provided significant information for structure-based vaccine and drug design. In this review, we summarized the research progress on the structures of ZIKV and its component proteins. We analyzed the structure identity and the differences between ZIKV and other flaviviruses. This information is crucial to guiding structure-based anti-ZIKV inhibitors and vaccine discovery.