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...

Many viruses go through a maturation step in the final stages of assembly before being transmitted to another host. The maturation process of flaviviruses is directed by the proteolytic cleavage of the precursor membrane protein (prM), turning inert virus into infectious particles. We have determined the 2.2 angstrom resolution crystal structure of a recombinant protein in which the dengue virus prM is linked to the envelope glycoprotein E. The structure represents the prM-E heterodimer and fits well into the cryo-electron microscopy density of immature virus at neutral pH. The pr peptide beta-barrel structure covers the fusion loop in E, preventing fusion with host cell membranes. The structure provides a basis for identifying the stages of its pH-directed conformational metamorphosis during maturation, ending with release of pr when budding from the host.

The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

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.

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 antibodydependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.

Enveloped viruses enter cells by inducing fusion of viral and cellular membranes, a process catalyzed by a specialized membrane-fusion protein expressed on their surface. This review focuses on recent structural studies of viral fusion proteins with an emphasis on their metastable prefusion form and on interactions with neutralizing antibodies. The fusion glycoproteins have been difficult to study because they are present in a labile, metastable form at the surface of infectious virions. Such metastability is a functional requirement, allowing these proteins to refold into a lower energy conformation while transferring the difference in energy to catalyze the membrane fusion reaction. Structural studies have shown that stable immunogens presenting the same antigenic sites as the labile wild-type proteins efficiently elicit potently neutralizing antibodies, providing a framework with which to engineer the antigens for stability, as well as identifying key vulnerability sites that can be used in next-generation subunit vaccine design.

The mechanism of action of a serotype-specific natural human antibody against dengue virus has been identified.

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...