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 cryoelectron 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 cryoelectron 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.Zika virus (ZIKV), a flavivirus, is thought to be principally transmitted to humans by the mosquito (Aedes aegypti) vector. Other flaviviriuses include West Nile virus (WNV), Japanese encephalitis virus (JEV), dengue virus (DENV) and yellow fever virus (YFV). ZIKV generally causes a mild disease. However, when pregnant women are infected with ZIKV, there is an increased risk of developing microcephaly in the fetus 1 .Retrospective analysis of data collected from a ZIKV outbreak in French Polynesia in 2013-2014 showed association of the virus with microcephaly 7 . Here we present the 3.7 Å resolution structure of ZIKV strain H/PF/2013 isolated during that outbreak 8 .For cryo-electron microscopy (cryoEM) studies, ZIKV was grown in the mosquito cell line at 28 °C and purified at 4 °C by polyethylene glycol precipitation, a sucrose cushion, followed by a potassium tartrate gradient. The gel analysis of the purified sample suggested it contained mostly mature virus (Extended Data Fig. 1). The ZIKV samples were incubated at 28 °C, 37 °C and 40 °C (mimicking high fever) for 30 min, before imaging by cryoEM (Fig. 1a). At 28 °C, there were broken and shrivelled particles together with some smooth surfaced particles (about 500 Å in diameter), similar to the compact DENV mature particles. Conversely, samples incubated at 37 °C and 40 °C showed many more smooth surfaced particles. The presence of a larger fraction of shrivelled particles at 28 °C could be due to the exposure of particles to high osmolality during purification. We speculate that ZIKV particles may expand into smooth surfaced particles when incubated at higher temperatures, making the lipid envelope more fluid, and allowing the structure to revert to its normal state. Some strains of DENV2 (New G...

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.

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. Dengue virus (DENV) is a mosquito-borne pathogen and the causative agent of dengue fever, dengue hemorrhagic fever (DHF), and the life-threatening dengue shock syndrome (DSS). Currently, DENV infects about 50 to 100 million people per year, resulting in 250,000 to 500,000 cases of DHF or DSS, making it a major health, social, and economic problem (1). This virus belongs to the family Flaviviridae, which also includes other major human pathogens, such as yellow fever virus, West Nile virus (WNV), tick-borne encephalitis virus, Japanese encephalitis virus, etc. The four DENV serotypes share high genetic homology, varying in amino acid sequence by about 25 to 40%. Genotypes within a serotype are even more conserved, containing about only 3% variation in amino acid sequence (2, 3).Envelope (E) protein is the major antigenic structure on the surface of DENV (4). Crystal structures of the ectodomain of the E protein showed that it consists of three domains (I, II, and III) and that E proteins likely exist as dimers in solution (5-7). Domain III participates in receptor binding, while domain II facilitates virus fusion via the interaction of its fusion loop at the tip of the domain with the endosomal membrane during virus entry into the cell (4). Cryo-electron microscopy (cryo-EM) structures (8, 9) of the mature DENV showed that the surface of the virus is made from 180 copies of E and 180 copies of membrane (M) proteins that are arranged in an icosahedral manner. There are three individual E proteins in each of the 60 asymmetric units (molecules A, B and C; also shown in Fig. 5B) (8, 9). Each of these E proteins is located close to one of the 2-, 3-, or 5-fold vertices, thereby having a different local chemical environment. The E proteins are organized as 90 head-to-tail homodimers. Three of these homodimers lie parallel to each other, forming a raft, and together with the other 29 rafts, they form a herringbone pattern on the surface of the virus.The previously published cryo-EM structures were done with DENV grown in mosquito cell lines incubated at 28 to 30°C for several days and then kept at 4°C during virus purification pr...

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. Dengue virus (DENV), the cause of dengue fever, infects 100 million people worldwide every year. It is a member of the Flaviviridae family (1), with four serotypes: DENV1, -2, -3, and -4. Infection with DENV usually causes a self-limiting fever accompanied by rashes and joint pain in patients but might lead to dengue hemorrhagic fever and dengue shock syndrome, which may be fatal.DENV consists of an ϳ500-Å-diameter protein shell embedded in a host-derived lipid membrane and encapsidates an 11-kb single-stranded positive-sense RNA genome. The dengue genome encodes three structural proteins, the core (or capsid), the premembrane (prM), and the envelope (E) protein, that form the virus particle, as well as seven nonstructural proteins (1) that are involved in replication of the virus genome. The newly synthesized immature DENV has a spiky appearance (2) (Fig. 1A) and is typically noninfectious unless it is complexed with certain antibodies (3). Virus maturation occurs during transportation of the virus particle through the trans-Golgi component network (TGN). The acidic environment of these compartments induces structural rearrangement of the virus surface proteins. During this initial maturation process, the furin protease cleaves prM molecules on the virus into pr and M. After leaving the cell, the cleaved pr dissociates from the virus surface, resulting in smooth, fully mature infectious virus particles (4) (Fig. 1B).The E protein is the major structural component of the viral surface. The ectodomain of E protein (5) contains three distinct domains, DI, DII, and DIII (also shown in Fig. 1C), which are connected by flexible links that allow rearrangement of domains during virus assembly, maturation, and infection (5-7). DIII is involved in attachment to host cell receptors, whereas DII is responsible for fusion to the host endosomal membrane during infection (8). The ectodomain is connected to the stem made from amphipathic helices ␣1 and ␣2. The stem, in turn, is anchored to the virus lipid membrane by two transmembrane (TM) ␣-helices, TM1 and TM2.The crystal structure of an E-prM complex (6) shows that pr has a ␤-barrel fold and caps the fusion loop of the E protein, consistent with its function in preventing the newly synthesized virus from fusing back into the cell during maturation. The furin cleavage site lies between the pr molecule and the ectodomain of M protein, which exists as a linear polypeptide ...

Dengue virus (DENV) infects ~400 million people annually. There is no licensed vaccine or therapeutic drug. Only a small fraction of the total DENV-specific antibodies in a naturally occurring dengue infection consists of highly neutralizing antibodies. Here we show that the DENV-specific human monoclonal antibody 5J7 is exceptionally potent, neutralizing 50% of virus at nanogram-range antibody concentration. The 9 Å resolution cryo-electron microscopy structure of the Fab 5J7–DENV complex shows that a single Fab molecule binds across three envelope proteins and engages three functionally important domains, each from a different envelope protein. These domains are critical for receptor binding and fusion to the endosomal membrane. The ability to bind to multiple domains allows the antibody to fully coat the virus surface with only 60 copies of Fab, that is, half the amount compared with other potent antibodies. Our study reveals a highly efficient and unusual mechanism of molecular recognition by an antibody.

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.Subject Categories Microbiology, Virology & Host Pathogen Interaction; Immunology

SUMMARY Zika virus (ZIKV), a mosquito-borne flavivirus, causes devastating congenital birth defects. We isolated a human monoclonal antibody (mAb), ZKA190, that potently cross-neutralizes multi-lineage ZIKV strains. ZKA190 is highly effective in vivo in preventing morbidity and mortality of ZIKV-infected mice. NMR and cryo-electron microscopy show its binding to an exposed epitope on DIII of the E protein. ZKA190 Fab binds all 180 E protein copies, altering the virus quaternary arrangement and surface curvature. However, ZIKV escape mutants emerged in vitro and in vivo in the presence of ZKA190, as well as of other neutralizing mAbs. To counter this problem, we developed a bispecific antibody (FIT-1) comprising ZKA190 and a second mAb specific for DII of E protein. In addition to retaining high in vitro and in vivo potencies, FIT-1 robustly prevented viral escape, warranting its development as a ZIKV immunotherapy.

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.