Flavivirus Nonstructural Protein Research Articles

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 β-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.

Japanese encephalitis virus NS1 and NS1' protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy.

Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1' is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1' can disrupt the blood-brain barrier (BBB) of mice, with NS1' exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1' protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1' protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1' treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1' on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism.IMPORTANCEJapanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1' protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1' of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1' is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1' can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1' in the disruption of BBB, thereby providing the potential therapeutic target for JE.

Secreted NS1 proteins of tick-borne encephalitis virus and West Nile virus block dendritic cell activation and effector functions.

The flavivirus non-structural protein 1 (NS1) is secreted from infected cells into the circulation and the serum levels correlate with disease severity. The effect of secreted NS1 (sNS1) on non-infected mammalian immune cells is largely unknown. Here, we expressed recombinant sNS1 proteins of tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) and investigated their effects on dendritic cell (DC) effector functions. Murine bone marrow-derived DCs (BMDCs) showed reduced surface expression of co-stimulatory molecules and decreased release of pro-inflammatory cytokines when treated with sNS1 of TBEV or WNV prior to poly(I:C) stimulation. Transcriptional profiles of BMDCs that were sNS1-exposed prior to poly(I:C) stimulation showed two gene clusters that were downregulated by TBEV or WNV sNS1 and that were associated with innate and adaptive immune responses. Functionally, both sNS1 proteins modulated the capacity for BMDCs to induce specific T-cell responses as indicated by reduced IFN-γ levels in both CD4+ and CD8+ T cells after BMDC co-cultivation. In human monocyte-derived DCs, poly(I:C)-induced upregulation of co-stimulatory molecules and cytokine responses were even more strongly impaired by TBEV sNS1 or WNV sNS1 pretreatment than in the murine system. Our findings indicate that exogenous flaviviral sNS1 proteins interfere with DC-mediated stimulation of T cells, which is crucial for the initiation of cell-mediated adaptive immune responses in human flavivirus infections. Collectively, our data determine soluble flaviviral NS1 as a virulence factor responsible for a dampened immune response to flavivirus infections. IMPORTANCE The effective initiation of protective host immune responses controls the outcome of infection, and dysfunctional T-cell responses have previously been associated with symptomatic human flavivirus infections. We demonstrate that secreted flavivirus NS1 proteins modulate innate immune responses of uninfected bystander cells. In particular, sNS1 markedly reduced the capacity of dendritic cells to stimulate T-cell responses upon activation. Hence, by modulating cellular host responses that are required for effective antigen presentation and initiation of adaptive immunity, sNS1 proteins may contribute to severe outcomes of flavivirus disease.

Secretory pathways and multiple functions of nonstructural protein 1 in flavivirus infection.

The genus Flavivirus contains a wide variety of viruses that cause severe disease in humans, including dengue virus, yellow fever virus, Zika virus, West Nile virus, Japanese encephalitis virus and tick-borne encephalitis virus. Nonstructural protein 1 (NS1) is a glycoprotein that encodes a 352-amino-acid polypeptide and has a molecular weight of 46-55 kDa depending on its glycosylation status. NS1 is highly conserved among multiple flaviviruses and occurs in distinct forms, including a dimeric form within the endoplasmic reticulum, a cell-associated form on the plasma membrane, or a secreted hexameric form (sNS1) trafficked to the extracellular matrix. Intracellular dimeric NS1 interacts with other NSs to participate in viral replication and virion maturation, while extracellular sNS1 plays a critical role in immune evasion, flavivirus pathogenesis and interactions with natural vectors. In this review, we provide an overview of recent research progress on flavivirus NS1, including research on the structural details, the secretory pathways in mammalian and mosquito cells and the multiple functions in viral replication, immune evasion, pathogenesis and interaction with natural hosts, drawing together the previous data to determine the properties of this protein.

Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells.

Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and β-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and β-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and β-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the β-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.

Flavivirus NS1 Triggers Tissue-Specific Disassembly of Intercellular Junctions Leading to Barrier Dysfunction and Vascular Leak in a GSK-3β-Dependent Manner.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins β-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of β-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of β-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3β (GSK-3β). Supporting this model, we found that a chemical inhibitor of GSK-3β reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3β as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Identification of Residues within Flavivirus Non-Structural Protein Associated with Mosquito Transmission

Flavivirus non-structural protein 1 (NS1) is present at high levels in patient blood and has been previously implicated in increasing virus acquisition by mosquito vectors, which may facilitate increased transmission. Therefore, flaviviruses transmitted by Aedes aegypti mosquitoes were hypothesised to have similarities within NS1, and potentially other NS proteins, that may correlate with mosquito-borne (rather than tick-borne or no vector) transmission. Representative flavivirus amino acid sequences were downloaded from the NCBI Protein database, aligned using viprbrc.org and then manually analysed to identify residues correlating with arthropod vector tropism. All available sequences for individual clinically important flaviviruses were then analysed on viprbrc.org to determine sequence conservation within viral species for those residues correlating with vector tropism. Overall, we found five residues in NS1 that were highly correlated with transmission by mosquitoes, had functionally relevant differences in amino acid side chain properties between mosquito- and tick-borne viruses and had conservation within viral species. Additionally, a further nine residues in NS3 and four residues in NS4A were identified using the same methodology. Importantly, for NS1, for which more data is available regarding its role in influencing transmission, the residues we identified have not been implicated in increased transmission thus far. Hence, future work would aim to test the impact of mutating these NS1 residues to elucidate whether they modulate NS1 secretion into the blood stream, virus replication and/or transmission. Gaining an understanding of the molecular determinants underpinning vector tropism could be useful in the creation of transmission-incompetent vectors.

Sulfated β-glucan from Agaricus subrufescens inhibits flavivirus infection and nonstructural protein 1-mediated pathogenesis

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1–6,1-3)-β-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 μg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 μg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 μg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

Isolation of alpaca-derived single-domain antibodies against zika virus non-structural proteins to interrogate viral replication

Abstract Zika virus (ZIKV) is a re-emergent flavivirus that triggered a global health emergency from 2015 to 2017. ZIKV and other impactful flaviviruses, including dengue virus, lack the antiviral treatments and vaccines needed to diminish their threat to communities world-wide. This is in part due to gaps in our understanding of the flavivirus lifecycle; despite numerous investigations into flavivirus non-structural (NS) protein function. I hope to forge a deeper understanding of crucial NS protein functions and interactions throughout the ZIKV replication cycle. I will use alpaca-derived variable heavy-chain-only (VHH) antibody fragments, isolated from an alpaca immunized with ZIKV NS proteins, to perturb the replication cycle and therefore identify key NS protein epitopes used during replication. I individually purified NS1, NS2b, NS3, NS4b, NS5 for use in alpaca immunization, phage display panning, and enzyme-linked immunosorbent assays (ELISAs). From the alpaca peripheral blood I isolated RNA and amplified VHH genes, enriched for NS-protein binders with iterative phage display, and screened potential high binders via ELISA. I have isolated VHH sequences that interact with the ZIKV helicase, NS3, and the RNA-dependent RNA polymerase, NS5. I plan to purify each VHH and express them in ZIKA-permissive A549 cells as stable lines. Using purified VHH, I will analyze their binding ability in-vitro by ELISA and bio layer interferometry, and quantify in-vitro inhibition of NS3 protease and helicase activity for NS3 binders, and NS5 polymerase and methyltransferase activity. With the stable cells lines I will investigate each VHHs ability to impede ZIKV virus production and formation of replication complexes in infected cell lines. Support from training grant "Interactions at the Microbe-Host Interface" T32AI007472

Role of the homologous MTase-RdRp interface of flavivirus intramolecular NS5 on duck tembusu virus

Flavivirus nonstructural protein 5 (NS5) harbors the N-terminal methyltransferase (MTase) and C-terminal polymerase RNA-dependent RNA polymerase (RdRp). The intramolecular NS5 features an integral MTase and RdRp interface with two components: a six-residue hydrophobic network and a GTR linker. Herein, the determinants of the MTase-RdRp interface and flavivirus substituted GTR linker were explored in TMUV replication and proliferation. First, the NanoLuc® Binary Technology (NanoBiT) and coimmunoprecipitation assays (Co-IP) methods confirmed the interaction between the MTase and RdRp domains of TMUV NS5. To screen for an optimal orientation for reporter gene fusion to the protein of interest, the signal activity of eight combinations of MTase and RdRp was explored. Intriguingly, all the combinations with the reporter gene fused to the C-terminal of MTase (1.1 C/2.1 C MTase) could barely detect any positive signal, suggesting a role for the GTR linker of the MTase C-terminal in MTase-RdRp affinity. Based on the flavivirus NS5 homologous interplay, we introduced alanine mutations into the MTase-RdRp interface of TMUV NS5. However, no single or pairwise mutation was found to abort the NS5 intramolecular interaction. Then, a mutated replicon and infectious clone were constructed to analyze the replication ability and properties of the recombinant virus. The mutant replicons of MTase F113A and M115A replicated to comparable extent as the wild type (WT). However, the replication level of the mutant MTase W121A was impaired without an obvious decrease in proliferation and virulence. Both the RdRp F351A and P585A mutants could replicate and proliferate well. Notably, the RdRp F467A virus was attenuated and did not strikingly impair the MTase-RdRp interaction. Furthermore, the TMUV was specifically compatible with the substituted NS5 with a Japanese encephalitis virus (JEV) GTR linker. Compensatory mutations were observed in the context of a defective MTase-RdRp interface after several passages of the rescued mutants in BHK-21 cells. A greater understanding of the molecular mechanism of the NS5 protein controlling duck TMUV replication will facilitate the design of novel therapies.

Structural Basis of Zika Virus Helicase in RNA Unwinding and ATP Hydrolysis.

The flavivirus nonstructural protein 3 helicase (NS3hel) is a multifunctional domain protein that is associated with DNA/RNA helicase, nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities. As an NTPase-dependent superfamily 2 (SF2) member, NS3hel employs an NTP-driven motor force to unwind double-stranded RNA while translocating along single-stranded RNA and is extensively involved in the viral replication process. Although the structures of SF2 helicases are widely investigated as promising drug targets, the mechanism of energy transduction between NTP hydrolysis and the RNA binding sites in ZIKV NS3hel remains elusive. Here, we report the crystal structure of ZIKV NS3hel in complex with its natural substrates ATP-Mn2+ and ssRNA. Distinct from other members of the Flavivirus genus, ssRNA binding to ZIKV NS3hel induces relocation of the active water molecules and ATP-associated metal ions in the NTP hydrolysis active site, which promotes the hydrolysis of ATP and the production of AMP. Our findings highlight the importance of the allosteric role of ssRNA on the modulation of ATP hydrolysis and energy utilization.

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication.

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3'UTR-U30 (5'-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3').

Flavivirus Persistence in Wildlife Populations.

A substantial number of humans are at risk for infection by vector-borne flaviviruses, resulting in considerable morbidity and mortality worldwide. These viruses also infect wildlife at a considerable rate, persistently cycling between ticks/mosquitoes and small mammals and reptiles and non-human primates and humans. Substantially increasing evidence of viral persistence in wildlife continues to be reported. In addition to in humans, viral persistence has been shown to establish in mammalian, reptile, arachnid, and mosquito systems, as well as insect cell lines. Although a considerable amount of research has centered on the potential roles of defective virus particles, autophagy and/or apoptosis-induced evasion of the immune response, and the precise mechanism of these features in flavivirus persistence have yet to be elucidated. In this review, we present findings that aid in understanding how vector-borne flavivirus persistence is established in wildlife. Research studies to be discussed include determining the critical roles universal flavivirus non-structural proteins played in flaviviral persistence, the advancement of animal models of viral persistence, and studying host factors that allow vector-borne flavivirus replication without destructive effects on infected cells. These findings underscore the viral–host relationships in wildlife animals and could be used to elucidate the underlying mechanisms responsible for the establishment of viral persistence in these animals.

Levels of Circulating NS1 Impact West Nile Virus Spread to the Brain.

Dengue virus (DENV) and West Nile virus (WNV) are arthropod-transmitted flaviviruses that cause systemic vascular leakage and encephalitis syndromes, respectively, in humans. However, the viral factors contributing to these specific clinical disorders are not completely understood. Flavivirus nonstructural protein 1 (NS1) is required for replication, expressed on the cell surface, and secreted as a soluble glycoprotein, reaching high levels in the blood of infected individuals. Extracellular DENV NS1 and WNV NS1 interact with host proteins and cells, have immune evasion functions, and promote endothelial dysfunction in a tissue-specific manner. To characterize how differences in DENV NS1 and WNV NS1 might function in pathogenesis, we generated WNV NS1 variants with substitutions corresponding to residues found in DENV NS1. We discovered that the substitution NS1-P101K led to reduced WNV infectivity in the brain and attenuated lethality in infected mice, although the virus replicated efficiently in cell culture and peripheral organs and bound at wild-type levels to brain endothelial cells and complement components. The P101K substitution resulted in reduced NS1 antigenemia in mice, and this was associated with reduced WNV spread to the brain. Because exogenous administration of NS1 protein rescued WNV brain infectivity in mice, we conclude that circulating WNV NS1 facilitates viral dissemination into the central nervous system and impacts disease outcomes. IMPORTANCE Flavivirus NS1 serves as an essential scaffolding molecule during virus replication but also is expressed on the cell surface and is secreted as a soluble glycoprotein that circulates in the blood of infected individuals. Although extracellular forms of NS1 are implicated in immune modulation and in promoting endothelial dysfunction at blood-tissue barriers, it has been challenging to study specific effects of NS1 on pathogenesis without disrupting its key role in virus replication. Here, we assessed WNV NS1 variants that do not affect virus replication and evaluated their effects on pathogenesis in mice. Our characterization of WNV NS1-P101K suggests that the levels of NS1 in the circulation facilitate WNV dissemination to the brain and affect disease outcomes. Our findings facilitate understanding of the role of NS1 during flavivirus infection and support antiviral strategies for targeting circulating forms of NS1.

Novel Nonnucleoside Inhibitors of Zika Virus Polymerase Identified through the Screening of an Open Library of Antikinetoplastid Compounds.

ABSTRACTZika virus (ZIKV) is a mosquito-borne pathogen responsible for neurological disorders (Guillain-Barré syndrome) and congenital malformations (microcephaly). Its ability to cause explosive epidemics, such as that of 2015 to 2016, urges the identification of effective antiviral drugs. Viral polymerase inhibitors constitute one of the most successful fields in antiviral research. Accordingly, the RNA-dependent RNA polymerase activity of flavivirus nonstructural protein 5 (NS5) provides a unique target for the development of direct antivirals with high specificity and low toxicity. Here, we describe the discovery and characterization of two novel nonnucleoside inhibitors of ZIKV polymerase. These inhibitors, TCMDC-143406 (compound 6) and TCMDC-143215 (compound 15) were identified through the screening of an open-resource library of antikinetoplastid compounds using a fluorescence-based polymerization assay based on ZIKV NS5. The two compounds inhibited ZIKV NS5 polymerase activity in vitro and ZIKV multiplication in cell culture (half-maximal effective concentrations [EC50] values of 0.5 and 2.6 μM for compounds 6 and 15, respectively). Both compounds also inhibited the replication of other pathogenic flaviviruses, namely, West Nile virus (WNV; EC50 values of 4.3 and 4.6 μM for compounds 6 and 15, respectively) and dengue virus 2 (DENV-2; EC50 values of 3.4 and 9.6 μM for compounds 6 and 15, respectively). Enzymatic assays confirmed that the polymerase inhibition was produced by a noncompetitive mechanism. Combinatorial assays revealed an antagonistic effect between both compounds, suggesting that they would bind to the same region of ZIKV polymerase. The nonnucleoside inhibitors of ZIKV polymerase here described could constitute promising lead compounds for the development of anti-ZIKV therapies and, eventually, broad-spectrum antiflavivirus drugs.

How NS1 Antibodies Prevent Severe Flavivirus Disease

The flavivirus genus consists of several major human pathogens including dengue (DENV) and Zika viruses. The flavivirus nonstructural protein 1 (NS1) plays an important role in disease progression, for example, in the development of severe dengue disease. Anti-NS1 antibodies have been shown to confer protection, and two new studies by Biering et al. and Modhiran et al. on the structure of NS1:antibody complexes reveal their mechanism of neutralization.

A broadly protective antibody that targets the flavivirus NS1 protein.

There are no approved flaviviral therapies and the development of vaccines against flaviruses has the potential of being undermined by antibody-dependent enhancement (ADE). The flavivirus nonstructural protein 1 (NS1) is a promising vaccine antigen with low ADE risk but has yet to be explored as a broad-spectrum therapeutic antibody target. Here, we provide the structural basis of NS1 antibody cross-reactivity through cocrystallization of the antibody 1G5.3 with NS1 proteins from dengue and Zika viruses. The 1G5.3 antibody blocks multi-flavivirus NS1-mediated cell permeability in disease-relevant cell lines, and therapeutic application of 1G5.3 reduces viremia and improves survival in dengue, Zika, and West Nile virus murine models. Finally, we demonstrate that 1G5.3 protection is independent of effector function, identifying the 1G5.3 epitope as a key site for broad-spectrum antiviral development.

Structural basis for antibody inhibition of flavivirus NS1-triggered endothelial dysfunction.

Medically important flaviviruses cause diverse disease pathologies and collectively are responsible for a major global disease burden. A contributing factor to pathogenesis is secreted flavivirus nonstructural protein 1 (NS1). Despite demonstrated protection by NS1-specific antibodies against lethal flavivirus challenge, the structural and mechanistic basis remains unknown. Here, we present three crystal structures of full-length dengue virus NS1 complexed with a flavivirus-cross-reactive, NS1-specific monoclonal antibody, 2B7, at resolutions between 2.89 and 3.96 angstroms. These structures reveal a protective mechanism by which two domains of NS1 are antagonized simultaneously. The NS1 wing domain mediates cell binding, whereas the β-ladder triggers downstream events, both of which are required for dengue, Zika, and West Nile virus NS1-mediated endothelial dysfunction. These observations provide a mechanistic explanation for 2B7 protection against NS1-induced pathology and demonstrate the potential of one antibody to treat infections by multiple flaviviruses.

Crystal structure of the NS3-like helicase from Alongshan virus.

Alongshan virus (ALSV) is an emerging human pathogen that was identified in China and rapidly spread to the European continent in 2019, raising concerns about public health. ALSV belongs to the distinct Jingmenvirus group within the Flaviviridae family with segmented RNA genomes. While segments 2 and 4 of the ALSV genome encode the VP1-VP3 proteins of unknown origin, segments 1 and 3 encode the NS2b-NS3 and NS5 proteins, which are related to Flavivirus nonstructural proteins, suggesting an evolutionary link between segmented and unsegmented viruses within the Flaviviridae family. Here, the enzymatic activity of the ALSV NS3-like helicase (NS3-Hel) was characterized and its crystal structure was determined to 2.9 Å resolution. ALSV NS3-Hel exhibits an ATPase activity that is comparable to those measured for Flavivirus NS3 helicases. The structure of ALSV NS3-Hel exhibits an overall fold similar to those of Flavivirus NS3 helicases. Despite the limited amino-acid sequence identity between ALSV NS3-Hel and Flavivirus NS3 helicases, structural features at the ATPase active site and the RNA-binding groove remain conserved in ALSV NS3-Hel. These findings provide a structural framework for drug design and suggest the possibility of developing a broad-spectrum antiviral drug against both Flavivirus and Jingmenvirus.

An attenuated Zika virus NS4B protein mutant is a potent inducer of antiviral immune responses

Live attenuated vaccines (LAVs) are one of the most important strategies to control flavivirus diseases. The flavivirus nonstructural (NS) 4B proteins are a critical component of both the virus replication complex and evasion of host innate immunity. Here we have used site-directed mutagenesis of residues in the highly conserved N-terminal and central hydrophobic regions of Zika virus (ZIKV) NS4B protein to identify candidate attenuating mutations. Three single-site mutants were generated, of which the NS4B-C100S mutant was more attenuated than the other two mutants (NS4B-C100A and NS4B-P36A) in two immunocompromised mouse models of fatal ZIKV disease. The ZIKV NS4B-C100S mutant triggered stronger type 1 interferons and interleukin-6 production, and higher ZIKV-specific CD4+ and CD8+ T-cell responses, but induced similar titers of neutralization antibodies compared with the parent wild-type ZIKV strain and a previously reported candidate ZIKV LAV with a 10-nucleotide deletion in 3′-UTR (ZIKV-3′UTR-Δ10). Vaccination with ZIKV NS4B-C100S protected mice from subsequent WT ZIKV challenge. Furthermore, either passive immunization with ZIKV NS4B-C100S immune sera or active immunization with ZIKV NS4B-C100S followed by the depletion of T cells affords full protection from lethal WT ZIKV challenge. In summary, our results suggest that the ZIKV NS4B-C100S mutant may serve as a candidate ZIKV LAV due to its attenuated phenotype and high immunogenicity.