Thiam Seng Ng
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gmsnts@nus.edu.sg
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Publication Near-atomic resolution cryo-electron microscopic structure of dengue serotype 4 virus(2014-01) Kostyuchenko, V.A.; Chew, P.L.; Ng, T.-S.; Lok, S.-M.; DUKE-NUS GRADUATE MEDICAL SCHOOL S'POREDengue virus (DENV), a mosquito-borne virus, is responsible for millions of cases of infections worldwide. There are four DENV serotypes (DENV1 to -4). After a primary DENV infection, the antibodies elicited confer lifetime protection against that DENV serotype. However, in a secondary infection with another serotype, the preexisting antibodies may cause antibody-dependent enhancement (ADE) of infection of macrophage cells, leading to the development of the more severe form of disease, dengue hemorrhagic fever. Thus, a safe vaccine should stimulate protection against all dengue serotypes simultaneously. To facilitate the development of a vaccine, good knowledge of different DENV serotype structures is crucial. Structures of DENV1 and DENV2 had been solved previously. Here we present a near-atomic resolution cryo-electron microscopy (cryo-EM) structure of mature DENV4. Comparison of the DENV4 structure with similar-resolution cryo-EM structures of DENV1 and DENV2 showed differences in surface charge distribution, which may explain their differences in binding to cellular receptors, such as heparin. Also, observed variations in amino acid residues involved in interactions between envelope and membrane proteins on the virus surface correlate with their ability to undergo structural changes at higher temperatures. © 2014, American Society for Microbiology.Publication Neutralization mechanism of a highly potent antibody against Zika virus(Nature Publishing Group, 2016) Zhang, S; Kostyuchenko, V.A; Ng, T.-S; Lim, X.-N; Ooi, J.S.G; Lambert, S; Tan, T.Y; Widman, D.G; Shi, J; Baric, R.S; Lok, S.-M; MICROBIOLOGY AND IMMUNOLOGY; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); BIOLOGICAL SCIENCES; DUKE-NUS MEDICAL SCHOOLThe rapid spread of Zika virus (ZIKV), which causes microcephaly and Guillain-Barré syndrome, signals an urgency to identify therapeutics. Recent efforts to rescreen dengue virus human antibodies for ZIKV cross-neutralization activity showed antibody C10 as one of the most potent. To investigate the ability of the antibody to block fusion, we determined the cryoEM structures of the C10-ZIKV complex at pH levels mimicking the extracellular (pH8.0), early (pH6.5) and late endosomal (pH5.0) environments. The 4.0 Å resolution pH8.0 complex structure shows that the antibody binds to E proteins residues at the intra-dimer interface, and the virus quaternary structure-dependent inter-dimer and inter-raft interfaces. At pH6.5, antibody C10 locks all virus surface E proteins, and at pH5.0, it locks the E protein raft structure, suggesting that it prevents the structural rearrangement of the E proteins during the fusion event - a vital step for infection. This suggests antibody C10 could be a good therapeutic candidate. © The Author(s) 2016.Publication A Human Antibody Neutralizes Different Flaviviruses by Using Different Mechanisms(Elsevier B.V., 2020) Zhang, S.; Loy, T.; Ng, T.-S.; Lim, X.-N.; Chew, S.-Y.V.; Tan, T.Y.; Xu, M.; Kostyuchenko, V.A.; Tukijan, F.; Shi, J.; Fink, K.; Lok, S.-M.; MICROBIOLOGY AND IMMUNOLOGY; BIOLOGICAL SCIENCES; DUKE-NUS MEDICAL SCHOOLHuman antibody SIgN-3C neutralizes dengue virus (DENV) and Zika virus (ZIKV) differently. DENV:SIgN-3C Fab and ZIKV:SIgN-3C Fab cryoelectron microscopy (cryo-EM) complex structures show Fabs crosslink E protein dimers at extracellular pH 8.0 condition and also when further incubated at acidic endosomal conditions (pH 8.0–6.5). We observe Fab binding to DENV (pH 8.0–5.0) prevents virus fusion, and the number of bound Fabs increase (from 120 to 180). For ZIKV, although there are already 180 copies of Fab at pH 8.0, virus structural changes at pH 5.0 are not inhibited. The immunoglobulin G (IgG):DENV structure at pH 8.0 shows both Fab arms bind to epitopes around the 2-fold vertex. On ZIKV, an additional Fab around the 5-fold vertex at pH 8.0 suggests one IgG arm would engage with an epitope, although the other may bind to other viruses, causing aggregation. For DENV2 at pH 5.0, a similar scenario would occur, suggesting DENV2:IgG complex would aggregate in the endosome. Hence, a single antibody employs different neutralization mechanisms against different flaviviruses. Zhang et al. show that a human monoclonal antibody SIgN-3C can neutralize closely related dengue and Zika virus via different mechanisms. The antibody neutralizes dengue virus by preventing virus:endosomal membrane fusion, although it aggregates Zika virus particles extracellularly. © 2020 The Author(s)Publication Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography(2013-04-02) Veesler, D.; Ng, T.-S.; Sendamarai, A.K.; Eilers, B.J.; Lawrence, C.M.; Lok, S.-M.; Young, M.J.; Johnson, J.E.; Fu, C.-Y.; DUKE-NUS GRADUATE MEDICAL SCHOOL S'PORESulfolobus turreted icosahedral virus (STIV) was isolated in acidic hot springs where it infects the archeon Sulfolobus solfataricus. We determined the STIV structure using near-atomic resolution electron microscopy and X-ray crystallography allowing tracing of structural polypeptide chains and visualization of transmembrane proteins embedded in the viral membrane. We propose that the vertex complexes orchestrate virion assembly by coordinating interactions of the membrane and various protein components involved. STIV shares the same coat subunit and penton base protein folds as some eukaryotic and bacterial viruses, suggesting that they derive from a common ancestor predating the divergence of the three kingdoms of life. One architectural motif (β-jelly roll fold) forms virtually the entire capsid (distributed in three different gene products), indicating that a single ancestral protein module may have been at the origin of its evolution.Publication Antibody affinity versus dengue morphology influences neutralization(Public Library of Science, 2021-02-23) Fibriansah, Guntur; Lim, Elisa X. Y.; Marzinek, Jan K.; Ng, Thiam-Seng; Tan, Joanne L.; Huber, Roland G.; Lim, Xin-Ni; Chew, Valerie S. Y.; Kostyuchenko, Victor A.; Shi, Jian; Anand, Ganesh S.; Bond, Peter J.; Crowe, James E.; Lok, Shee-Mei; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); BIOLOGICAL SCIENCES; DUKE-NUS MEDICAL SCHOOLDifferent strains within a dengue serotype (DENV1-4) can have smooth, or “bumpy” surface morphologies with different antigenic characteristics at average body temperature (37C). We determined the neutralizing properties of a serotype cross-reactive human monoclonal antibody (HMAb) 1C19 for strains with differing morphologies within the DENV1 and DENV2 serotypes. We mapped the 1C19 epitope to E protein domain II by hydrogen deuterium exchange mass spectrometry, cryoEM and molecular dynamics simulations, revealing that this epitope is likely partially hidden on the virus surface. We showed the antibody has high affinity for binding to recombinant DENV1 E proteins compared to those of DENV2, consistent with its strong neutralizing activities for all DENV1 strains tested regardless of their morphologies. This finding suggests that the antibody could out-compete E-to-E interaction for binding to its epitope. In contrast, for DENV2, HMAb 1C19 can only neutralize when the epitope becomes exposed on the bumpy-surfaced particle. Although HMAb 1C19 is not a suitable therapeutic candidate, this study with HMAb 1C19 shows the importance of choosing a high-affinity antibody that could neutralize diverse dengue virus morphologies for therapeutic purposes. Copyright: © 2021 Fibriansah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Publication Molecular basis of dengue virus serotype 2 morphological switch from 29°C to 37°C(Public Library of Science, 2019) Lim, X.-N.; Shan, C.; Marzinek, J.K.; Dong, H.; Ng, T.S.; Ooi, J.S.G.; Fibriansah, G.; Wang, J.; Verma, C.S.; Bond, P.J.; Shi, P.-Y.; Lok, S.-M.; DEAN'S OFFICE (DUKE-NUS MEDICAL SCHOOL); BIOLOGICAL SCIENCES; DUKE-NUS MEDICAL SCHOOLThe ability of DENV2 to display different morphologies (hence different antigenic properties) complicates vaccine and therapeutics development. Previous studies showed most strains of laboratory adapted DENV2 particles changed from smooth to "bumpy" surfaced morphology when the temperature is switched from 29°C at 37°C. Here we identified five envelope (E) protein residues different between two alternative passage history DENV2 NGC strains exhibiting smooth or bumpy surface morphologies. Several mutations performed on the smooth DENV2 infectious clone destabilized the surface, as observed by cryoEM. Molecular dynamics simulations demonstrated how chemically subtle substitution at various positions destabilized dimeric interactions between E proteins. In contrast, three out of four DENV2 clinical isolates showed a smooth surface morphology at 37°C, and only at high fever temperature (40°C) did they become "bumpy". These results imply vaccines should contain particles representing both morphologies. For prophylactic and therapeutic treatments, this study also informs on which types of antibodies should be used at different stages of an infection, i.e., those that bind to monomeric E proteins on the bumpy surface or across multiple E proteins on the smooth surfaced virus. Copyright © 2019 Lim et al.Publication A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface(2014) Fibriansah G.; Tan J.L.; Smith S.A.; de Alwis A.R.; Ng T.-S.; Kostyuchenko V.A.; Ibarra K.D.; Wang J.; Harris E.; de Silva A.; Crowe J.E.; Lok S.-M.; DUKE-NUS MEDICAL SCHOOLDengue 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. © 2014 The Authors.Publication High flavivirus structural plasticity demonstrated by a non-spherical morphological variant(Nature Research, 2020-06-19) Morrone, S.R.; Chew, V.S.Y.; Lim, X.-N.; Ng, T.-S.; Kostyuchenko, V.A.; Zhang, S.; Chew, P.-L.; Lee, J.; Tan, J.L.; Wang, J.; Tan, T.Y.; Shi, J.; Screaton, G.; Morais, M.C.; Lok, S.-M.; MICROBIOLOGY AND IMMUNOLOGY; BIOLOGICAL SCIENCES; DUKE-NUS MEDICAL SCHOOLPrevious 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. © 2020, The Author(s).Publication Structural changes in dengue virus when exposed to a temperature of 37°C(2013-07) Fibriansah, G.; Ng, T.-S.; Kostyuchenko, V.A.; Lee, J.; Lee, S.; Wang, J.; Lok, S.-M.; DUKE-NUS GRADUATE MEDICAL SCHOOL S'POREPrevious binding studies of antibodies that recognized a partially or fully hidden epitope suggest that insect cell-derived dengue virus undergoes structural changes at an elevated temperature. This was confirmed by our cryo-electron microscopy images of dengue virus incubated at 37°C, where viruses change their surface from smooth to rough. Here we present the cryo-electron microscopy structures of dengue virus at 37°C. Image analysis showed four classes of particles. The three-dimensional (3D) map of one of these classes, representing half of the imaged virus population, shows that the E protein shell has expanded and there is a hole at the 3-fold vertices. Fitting E protein structures into the map suggests that all of the interdimeric and some intradimeric E protein interactions are weakened. The accessibility of some previously found cryptic epitopes on this class of particles is discussed. © 2013, American Society for Microbiology.Publication Immature and mature dengue serotype 1 virus structures provide insight into the maturation process(2013-07) Kostyuchenko, V.A.; Zhang, Q.; Tan, J.L.; Ng, T.-S.; Lok, S.-M.; DUKE-NUS GRADUATE MEDICAL SCHOOL S'POREDengue virus is a major human pathogen that has four serotypes (DENV1 to -4). Here we report the cryoelectron microscopy (cryo-EM) structures of immature and mature DENV1 at 6- and 4.5-Å resolution, respectively. The subnanometer-resolution maps allow accurate placement of all of the surface proteins. Although the immature and mature viruses showed vastly different surface protein organizations, the envelope protein transmembrane (E-TM) regions remain in similar positions. The pivotal role of the E-TM regions leads to the identification of the start and end positions of all surface proteins during maturation. © 2013, American Society for Microbiology.