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Dengue virus (DENV), which consists of four serotypes (DENV1‐4), infects over 400 million people annually. Previous studies have indicated most human monoclonal antibodies (HMAbs) from dengue patients are cross‐reactive and poorly neutralizing. Rare neutralizing HMAbs are usually serotype‐specific and bind to quaternary structure‐dependent epitopes. We determined the structure of DENV1 complexed with Fab fragments of a highly potent HMAb 1F4 to 6 Å resolution by cryo‐EM. Although HMAb 1F4 appeared to bind to virus and not E proteins in ELISAs in the previous study, our structure showed that the epitope is located within an envelope (E) protein monomer, and not across neighboring E proteins. The Fab molecules bind to domain I (DI), and DI‐DII hinge of the E protein. We also showed that HMAb 1F4 can neutralize DENV at different stages of viral entry in a cell type and receptor dependent manner. The structure reveals the mechanism by which this potent and specific antibody blocks viral infection. Synopsis Vaccine development against Dengue disease is complicated by the virus serotype priming to secondary infections. Structure of a potent human antibody 1F4 complexed with DENV serotype 1 highlights the hinge angle of the viral protein as critical for vaccine design. DI‐DII hinge is a common epitope recognised by highly neutralising human antibodies. The conformation angle of the hinge is critical for 1F4 binding. 1F4 recognises E protein monomers on the virus surface but not recombinant E proteins due to the conserved hinge angle of endogenous viral E proteins. Graphical Abstract Vaccine development against Dengue disease is complicated by the virus serotype priming to secondary infections. Structure of a potent human antibody 1F4 complexed with DENV serotype 1 highlights the hinge angle of the viral protein as critical for vaccine design.

The 3.7 Å cryo-electron microscopy structure of Zika virus is presented, revealing a typical flavivirus architecture; in contrast to the related flavivirus dengue virus, Zika virus is thermally stable at 40 °C, and this structural stability may be a feature that helps it to survive in semen, saliva and urine. What makes Zika virus tough Shee-Mei Lok and colleagues provide a 3.7 Å cryo-electron microscopy view of Zika virus, revealing typical flavivirus architecture. They report that in contrast to the related flavivirus dengue virus, Zika virus is thermally stable at 40 °C, and speculate that this structural stability may contribute to the ability of the virus to survive in semen, saliva and urine. Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses 1 , and with Guillian–Barré syndrome in adults 2 . Here we present the 3.7 Å resolution cryo-electron microscopy structure of ZIKV, and show that the overall architecture of the virus is similar to that of other flaviviruses. Sequence and structural comparisons of the ZIKV envelope (E) protein with other flaviviruses show that parts of the E protein closely resemble the neurovirulent West Nile and Japanese encephalitis viruses, while others are similar to dengue virus (DENV). However, the contribution of the E protein to flavivirus pathobiology is currently not understood. The virus particle was observed to be structurally stable even when incubated at 40 °C, in sharp contrast to the less thermally stable DENV 3 . This is also reflected in the infectivity of ZIKV compared to DENV serotypes 2 and 4 (DENV2 and DENV4) at different temperatures. The cryo-electron microscopy structure shows a virus with a more compact surface. This structural stability of the virus may help it to survive in the harsh conditions of semen 4 , saliva 5 and urine 6 . Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.

Previous flavivirus (dengue and Zika viruses) studies showed largely spherical particles either with smooth or bumpy surfaces. Here, we demonstrate flavivirus particles have high structural plasticity by the induction of a non-spherical morphology at elevated temperatures: the club-shaped particle (clubSP), which contains a cylindrical tail and a disc-like head. Complex formation of DENV and ZIKV with Fab C10 stabilize the viruses allowing cryoEM structural determination to ~10 Å resolution. The caterpillar-shaped (catSP) Fab C10:ZIKV complex shows Fabs locking the E protein raft structure containing three E dimers. However, compared to the original spherical structure, the rafts have rotated relative to each other. The helical tail structure of Fab C10:DENV3 clubSP showed although the Fab locked an E protein dimer, the dimers have shifted laterally. Morphological diversity, including clubSP and the previously identified bumpy and smooth-surfaced spherical particles, may help flavivirus survival and immune evasion. Dengue (DENV) and Zika (ZIKV) viruses normally display as smooth spherical particles, while DENV can also become bumpy-surfaced, resulting in immune evasion. Here, Morrone et al. report DENV and ZIKV infectious club-shaped particles (clubSP) that display distinct antibody binding properties.

Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X‐ray crystallography we have defined the structure of the flavivirus cross‐reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo‐electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion‐loop‐specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion‐loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross‐reactive antibodies are often weakly neutralizing they also may contribute to antibody‐dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

About the Authors: Ter Yong Tan Affiliations Programme in Emerging Infectious Diseases, Duke–National University of Singapore Medical School, Singapore, Singapore, Centre for BioImaging Sciences, Department of Biological Sciences, National University of Singapore, Singapore, Singapore ORCID logo http://orcid.org/0000-0002-6540-7026 Guntur Fibriansah Affiliations Programme in Emerging Infectious Diseases, Duke–National University of Singapore Medical School, Singapore, Singapore, Centre for BioImaging Sciences, Department of Biological Sciences, National University of Singapore, Singapore, Singapore ORCID logo http://orcid.org/0000-0002-9990-3335 Shee-Mei Lok * E-mail: sheemei.lok@duke-nus.edu.sg Affiliations Programme in Emerging Infectious Diseases, Duke–National University of Singapore Medical School, Singapore, Singapore, Centre for BioImaging Sciences, Department of Biological Sciences, National University of Singapore, Singapore, Singapore ORCID logo http://orcid.org/0000-0003-4631-8041 Citation: Tan TY, Fibriansah G, Lok S-M (2020) Capsid protein is central to the birth of flavivirus particles. C, Capsid; cryoEM, cryo-electron microscopy; E, Envelope; ER, endoplasmic reticulum; prM, precursor Membrane; TGN, trans-Golgi network; TM, transmembrane. https://doi.org/10.1371/journal.ppat.1008542.g001 [Figure omitted. Three neighboring building blocks are shown and colored in orange, purple, and brown. asu, asymmetric unit; C, Capsid; cryoEM, cryo-electron microscopy; E, Envelope; Fab, antigen-binding fragment; ImmZIKV, immature ZIKV; prM, precursor Membrane; ZIKV, Zika virus. https://doi.org/10.1371/journal.ppat.1008542.g002 The surface protein structures prM and E of the immature and mature flavivirus particles Cryo-electron microscopy (cryoEM) structures of the immature and mature flavivirus show that their surface proteins, although they are organized in a vastly different way, both exhibit icosahedral symmetries (Fig 2A) [2–4]. During the egress of the immature virus through the acidic compartments of the trans-Golgi network, the low pH triggers the reorganization of the heterotrimeric spikes into a dimeric surface protein organization (Figs 1A–1C and 2A) [5].

Structures of flavivirus (dengue virus and Zika virus) particles are known to near-atomic resolution and show detailed structure and arrangement of their surface proteins (E and prM in immature virus or M in mature virus). By contrast, the arrangement of the capsid proteins:RNA complex, which forms the core of the particle, is poorly understood, likely due to inherent dynamics. Here, we stabilize immature Zika virus via an antibody that binds across the E and prM proteins, resulting in a subnanometer resolution structure of capsid proteins within the virus particle. Fitting of the capsid protein into densities shows the presence of a helix previously thought to be removed via proteolysis. This structure illuminates capsid protein quaternary organization, including its orientation relative to the lipid membrane and the genomic RNA, and its interactions with the transmembrane regions of the surface proteins. Results show the capsid protein plays a central role in the flavivirus assembly process. The structure of flavivirus surface proteins has been elucidated, but the conformation of capsid proteins within particles is less clear. Here, the authors provide a subnanometer resolution structure of Zika virus capsid protein within the virus particle, elucidating its quaternary organization and role in flavivirus packaging.

Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional. The intracellular dimeric NS1 (iNS1) forms part of the viral replication complex. Previous studies suggest the extracellular secreted NS1 (sNS1), which is a major factor contributing to DHF, exists as hexamers. The structure of the iNS1 is well-characterised but not that of sNS1. Here we show by cryoEM that the recombinant sNS1 exists in multiple oligomeric states: the tetrameric (stable and loose conformation) and hexameric structures. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain – elongated β-sheet or β-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics. The extracellular secreted NS1 (sNS1) is a major factor contributing to dengue hemorrhagic fever. Here, Shu et al. report sNS1 exists in multiple oligomeric states and presents a tetrameric structure.

An antibody to dengue virus that lies flat on its target, neutralizes the virus and also prevents antibody-dependent enhancement of infection is now identified.

There are four closely-related dengue virus (DENV) serotypes. Infection with one serotype generates antibodies that may cross-react and enhance infection with other serotypes in a secondary infection. We demonstrated that DENV serotype 2 (DENV2)–specific human monoclonal antibody (HMAb) 2D22 is therapeutic in a mouse model of antibody-enhanced severe dengue disease. We determined the cryo–electron microscopy (cryo-EM) structures of HMAb 2D22 complexed with two different DENV2 strains. HMAb 2D22 binds across viral envelope (E) proteins in the dimeric structure, which probably blocks the E protein reorganization required for virus fusion. HMAb 2D22 \"locks\" two-thirds of or all dimers on the virus surface, depending on the strain, but neutralizes these DENV2 strains with equal potency. The epitope defined by HMAb 2D22 is a potential target for vaccines and therapeutics.

Dengue secreted non-structural protein 1 (sNS1) contributes to the vascular permeability symptom of severe dengue hemorrhagic fever. Previous flavivirus sNS1 structures suggest that they predominantly exist as loose tetramers. Here, we report two stable tetramer structures (3.1–3.6 Å) together with loose tetramers. Formation of the stable tetramers involves a dramatic rearrangement of their N-terminal regions compared to the loose tetramers. We observe a higher molecular weight complex (HMWC) comprising dimeric sNS1 and heat shock proteins, which exhibits much lower endothelial hyperpermeability activity than the tetramer-enriched samples. We also determine high-resolution structures of the sNS1 complex with heparin, an analogue of the attachment factor heparan sulfate, showing that heparin binds to a conserved basic groove on the outer surface of the dimer, at the intra-dimer interface. Pre-incubation of sNS1 with heparin reduces its endothelial hyperpermeability activity. Our findings provide important structural information for future sNS1-based drug or vaccine design. Dengue virus sNS1 is a virulence factor in severe dengue. Cryo-EM structures reveal multiple sNS1 oligomeric states and define a heparan sulfate–binding groove. Tetrameric NS1 is identified as the functional unit inducing endothelial permeability.