Antibody affinity versus dengue morphology influences neutralization.

Different strains within a dengue serotype (DENV1-4) can have smooth, or "bumpy" surface morphologies with different antigenic characteristics at average body temperature (37°C). 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.

Structural basis of Chikungunya virus inhibition by monoclonal antibodies.

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Structure of the thermally stable Zika virus.

Zika virus (ZIKV), formerly a neglected pathogen, has recently been associated with microcephaly in fetuses, and with Guillian-Barré syndrome in adults. 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. 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, saliva and urine. Antibodies or drugs that destabilize the structure may help to reduce the disease outcome or limit the spread of the virus.

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase, an enzyme essential for the alleviation of oxidative stress.

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H2O2. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC1 -172 and EcAhpC1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC1 -172 and EcAhpC1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC-AhpF assembly has been demonstrated.

Near-atomic resolution cryo-electron microscopic structure of dengue serotype 4 virus.

Dengue 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.

Structural changes in dengue virus when exposed to a temperature of 37°C.

Previous 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.

Immature and mature dengue serotype 1 virus structures provide insight into the maturation process.

Dengue 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.

The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria.

The contractile tail of bacteriophage T4 is a molecular machine that facilitates very high viral infection efficiency. Its major component is a tail sheath, which contracts during infection to less than half of its initial length. The sheath consists of 138 copies of the tail sheath protein, gene product (gp) 18, which surrounds the central non-contractile tail tube. The contraction of the sheath drives the tail tube through the outer membrane, creating a channel for the viral genome delivery. A crystal structure of about three quarters of gp18 has been determined and was fitted into cryo-electron microscopy reconstructions of the tail sheath before and after contraction. It was shown that during contraction, gp18 subunits slide over each other with no apparent change in their structure.

Structural basis for the preferential recognition of immature flaviviruses by a fusion-loop antibody.

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.

The structure of barmah forest virus as revealed by cryo-electron microscopy at a 6-angstrom resolution has detailed transmembrane protein architecture and interactions.

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design.

Structural studies of the giant mimivirus.

Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60 degrees . The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Angstroms.

An icosahedral algal virus has a complex unique vertex decorated by a spike.

Paramecium bursaria Chlorella virus-1 is an icosahedrally shaped, 1,900-A-diameter virus that infects unicellular eukaryotic green algae. A 5-fold symmetric, 3D reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. The pocket might contain enzymes for use in the initial stages of infection. The unique vertex consists of virally coded proteins, some of which have been identified. Comparison of shape, size, and location of the spike with similar features in bacteriophages T4 and P22 suggests that the spike might be a cell-puncturing device. Similar asymmetric features may have been missed in previous analyses of many other viruses that had been assumed to be perfectly icosahedral.

CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever.

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 ß-sheet or ß-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.

Morphogenesis of the T4 tail and tail fibers.

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study.

The tail structure of bacteriophage T4 and its mechanism of contraction.

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.

A Human Antibody Neutralizes Different Flaviviruses by Using Different Mechanisms.

Human 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.

Capsid protein structure in Zika virus reveals the flavivirus assembly process.

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.

High flavivirus structural plasticity demonstrated by a non-spherical morphological variant.

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.

Visualization of the externalized VP2 N termini of infectious human parvovirus B19.

The structures of infectious human parvovirus B19 and empty wild-type particles were determined by cryoelectron microscopy (cryoEM) to 7.5-A and 11.3-A resolution, respectively, assuming icosahedral symmetry. Both of these, DNA filled and empty, wild-type particles contain a few copies of the minor capsid protein VP1. Comparison of wild-type B19 with the crystal structure and cryoEM reconstruction of recombinant B19 particles consisting of only the major capsid protein VP2 showed structural differences in the vicinity of the icosahedral fivefold axes. Although the unique N-terminal region of VP1 could not be visualized in the icosahedrally averaged maps, the N terminus of VP2 was shown to be exposed on the viral surface adjacent to the fivefold beta-cylinder. The conserved glycine-rich region is positioned between two neighboring, fivefold-symmetrically related VP subunits and not in the fivefold channel as observed for other parvoviruses.

Mechanism of Enhanced Immature Dengue Virus Attachment to Endosomal Membrane Induced by prM Antibody.

Dengue virus (DENV) particles are released from cells in different maturation states. Fully immature DENV (immDENV) is generally non-infectious, but can become infectious when complexed with anti-precursor membrane (prM) protein antibodies. It is unknown how anti-prM antibody-coated particles can undergo membrane fusion since the prM caps the envelope (E) protein fusion loop. Here, we determined cryoelectron microscopy (cryo-EM) maps of the immDENV:anti-prM complex at different pH values, mimicking the extracellular (pH 8.0) or endosomal (pH 5.0) environments. At pH 5.0, there are two structural classes with fewer antibodies bound than at pH 8.0. These classes may represent different maturation states. Molecular simulations, together with the measured high-affinity pr:antibody interaction (versus the weak pr:E interaction) and also the low pH cryo-EM structures, suggest how antibody:pr complex can dislodge from the E protein at low pH. This exposes the E protein fusion loop enhancing virus interaction with endosomes.