Enterovirus D68 (EV-D68) is a member of Picornaviridae and is a causative agent of recent outbreaks in the USA of respiratory illness in children. We report here the crystal structures of EV-D68 and its complex with pleconaril, a capsid binding compound that had been developed as an anti-rhinovirus drug. The hydrophobic drug binding pocket in viral protein 1 contained density that is consistent with a fatty acid of about 10 carbon atoms. This density could be displaced by pleconaril. We also showed that pleconaril inhibits EV-D68 at a half maximal effective concentration (EC50) of 430 nM and might, therefore, be a possible drug candidate to alleviate EV-D68 outbreaks.

We report on a conformational transition of dengue virus when changing the temperature from that present in its mosquito vectors to that of its human host. Using cryoelectron microscopy, we show that although the virus has a smooth surface, a diameter of ∼500 Å, and little exposed membrane at room temperature, the virions have a bumpy appearance with a diameter of ∼550 Å and some exposed membrane at 37°C. The bumpy structure at 37°C was found to be similar to the previously predicted structure of an intermediate between the smooth mature and fusogenic forms. As humans have a body temperature of 37°C, the bumpy form of the virus would be the form present in humans. Thus, optimal dengue virus vaccines should induce antibodies that preferentially recognize epitopes exposed on the bumpy form of the virus.host temperature | irreversible and conformational change | temperature dependence | cryo-EM D engue virus (DENV), together with other significant arthropod-borne human pathogens such as West Nile, yellow fever, and Japanese encephalitis viruses, belongs to the flavivirus genus of the Flaviviridae family of RNA viruses (1, 2). The Flavivirus genus has been subdivided into the mosquito-borne viruses (e.g., dengue, yellow fever, and Japanese encephalitis), the tick-borne viruses, and those that do not use arthropod vectors (3). The four related DENV serotypes are transmitted by the Aedes aegypti and Aedes albopictus mosquitoes. The absence of a coordinated and comprehensive mosquito abatement programs has contributed to the global spread of the mosquito vectors and human DENV infection. DENV now infects ∼230 million people worldwide each year, with an estimated 3.6 billion people living in areas of risk (4). DENV infection can cause dengue fever, more severe dengue hemorrhagic fever (DHF), and life-threatening dengue shock syndrome (DSS) (5). Secondary infection with a heterologous DENV serotype increases the risk of developing DHF and DSS. Currently, there are neither licensed vaccines nor effective antiviral drugs against DENV. Indeed, new approaches to vaccine development may be needed, as the most advanced tetravalent live-attenuated DENV vaccine candidate showed a poor 30% overall efficacy rate in a recently published phase 2b clinical trial (6, 7).DENV has an 11-kb, positive-sense RNA genome that encodes a capsid protein, a precursor membrane glycoprotein, an enveloped glycoprotein (E), and seven nonstructural proteins (8). The X-ray crystallographic structure of the E protein (9-11) shows that it has three ectodomains (DI, DII, and DIII). Domain DII has a fusion loop at its distal tip and DIII has an Ig-like fold. The E ectodomain is anchored to the viral membrane by its C-terminal transmembrane region and its ∼50 amino acids amphipathic α-helical "stem" region (9,(12)(13)(14).The structure of mature DENV, propagated at ∼30°C, determined by combining cryoelectron microscopy (cryo-EM) of the whole virus (8, 15) with crystallography of the protein components (9-11, 16) shows 90 E dimers arranged in an icosahedral...

Human enterovirus D68 (EV-D68) is a causative agent of childhood respiratory diseases and has now emerged as a global public health threat. Nevertheless, knowledge of the tissue tropism and pathogenesis of EV-D68 has been hindered by a lack of studies on the receptor-mediated EV-D68 entry into host cells. Here we demonstrate that cell surface sialic acid is essential for EV-D68 to bind to and infect susceptible cells. Crystal structures of EV-D68 in complex with sialylated glycan receptor analogues show that they bind into the ‘canyon' on the virus surface. The sialic acid receptor induces a cascade of conformational changes in the virus to eject a fatty-acid-like molecule that regulates the stability of the virus. Thus, virus binding to a sialic acid receptor and to immunoglobulin-like receptors used by most other enteroviruses share a conserved mechanism for priming viral uncoating and facilitating cell entry.

Enterovirus D68 (EV-D68) is an emerging pathogen that can cause severe respiratory disease and is associated with cases of paralysis, especially among children. Heretofore, information on host factor requirements for EV-D68 infection is scarce. Haploid genetic screening is a powerful tool to reveal factors involved in the entry of pathogens. We performed a genome-wide haploid screen with the EV-D68 prototype Fermon strain to obtain a comprehensive overview of cellular factors supporting EV-D68 infection. We identified and confirmed several genes involved in sialic acid (Sia) biosynthesis, transport, and conjugation to be essential for infection. Moreover, by using knockout cell lines and gene reconstitution, we showed that both α2,6- and α2,3-linked Sia can be used as functional cellular EV-D68 receptors. Importantly, the screen did not reveal a specific protein receptor, suggesting that EV-D68 can use multiple redundant sialylated receptors. Upon testing recent clinical strains, we identified strains that showed a similar Sia dependency, whereas others could infect cells lacking surface Sia, indicating they can use an alternative, nonsialylated receptor. Nevertheless, these Sia-independent strains were still able to bind Sia on human erythrocytes, raising the possibility that these viruses can use multiple receptors. Sequence comparison of Sia-dependent and Sia-independent EV-D68 strains showed that many changes occurred near the canyon that might allow alternative receptor binding. Collectively, our findings provide insights into the identity of the EV-D68 receptor and suggest the possible existence of Sia-independent viruses, which are essential for understanding tropism and disease.

Flaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted "open" fusogenic trimer. IMPORTANCEThe structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an "open" conformation, which is distinct from the "closed" conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130°to the postfusion orientation and thus precludes the stem region from "zipping" together the three E proteins along the domain II boundaries into the "closed" postfusion conformation, thus inhibiting fusion. Enveloped viruses enter cells by fusing their lipid membrane with specific membranes of the host cell. In the endosome, this process is often triggered by the acidic environment, which promotes virion glycoproteins to form oligomeric (usually trimeric) structures in which each monomer within the oligomer has a hydrophobic peptide at its extremity. Trimeric fusogenic structures have been identified in numerous viruses that have been classified into at least three types based on the nature of the fusion peptide (1, 2). In many cases, the structure of the mature virus and the postfusion structure are known. Based on these results, different fusion mechanisms have been proposed (3-8). Although the prefusion fusogenic structures of some viruses can be created by low-pH or particular lipid environments, it has been difficult to define the prefusion fusogenic state of flaviviruses because of their instability and propensity to fuse with neighboring virions.Dengue virus (DENV) is a member of the Flaviviridae family of positive-stranded RNA viruses, which include arthropod-borne human pathogens such as West Nile, Japanese encephalitis, and yellow fever viruses. Each year, approximately 390 million people become infected by DENV, resulting in about 20,000 deaths (9). DENV infections cause a spectrum of clinical diseases ranging from acute dengue fever to severe, potentially fatal, dengue hemorrhagic fever and shock syndrome (10). ...

Enterovirus D68 (EV-D68) belongs to a group of enteroviruses that contain a single positive-sense RNA genome surrounded by an icosahedral capsid. Like common cold viruses, EV-D68 mainly causes respiratory infections and is acid-labile. The molecular mechanism by which the acid-sensitive EV-D68 virions uncoat and deliver their genome into a host cell is unknown. Using cryoelectron microscopy (cryo-EM), we have determined the structures of the full native virion and an uncoating intermediate [the A (altered) particle] of EV-D68 at 2.2- and 2.7-Å resolution, respectively. These structures showed that acid treatment of EV-D68 leads to particle expansion, externalization of the viral protein VP1 N termini from the capsid interior, and formation of pores around the icosahedral twofold axes through which the viral RNA can exit. Moreover, because of the low stability of EV-D68, cryo-EM analyses of a mixed population of particles at neutral pH and following acid treatment demonstrated the involvement of multiple structural intermediates during virus uncoating. Among these, a previously undescribed state, the expanded 1 (“E1”) particle, shows a majority of internal regions (e.g., the VP1 N termini) to be ordered as in the full native virion. Thus, the E1 particle acts as an intermediate in the transition from full native virions to A particles. Together, the present work delineates the pathway of EV-D68 uncoating and provides the molecular basis for the acid lability of EV-D68 and of the related common cold viruses.

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Exogenous 20-hydroxyeicosatetraenoic acid (20-HETE) increases the growth of human glioma cells in vitro. However, glioma cells in culture show negligible 20-HETE synthesis. We examined whether inducing the expression of a 20-HETE synthase in a human glioma U251 cell line would increase proliferation. U251 cells transfected with CYP4A1 cDNA (termed U251 O) increased the formation of 20-HETE from less than 1 to over 60 pmol/min/mg proteins and increased their proliferation rate by 2-fold (p Ͻ 0.01). Compared with control U251, U251 O cells were rounded, smaller, showed a disorganized cytoskeleton, exhibited reduced vinculin staining, and were easily detached from the growing surface. They showed a marked increase in dihydroethidium staining, suggesting increased oxidative stress. The expression of phosphorylated extracellular signal-regulated kinase 1/2, cyclin D1/2, and vascular endothelial growth factor was markedly elevated in U251 O. The hyperproliferative and signaling effects seen in U251 O cells are abolished by selective CYP4A inhibition of 20-HETE formation with HET0016 [N-hydroxy-NЈ-(4-butyl-2-methylphenyl)-formamidine], by small interfering RNA against the enzyme, and by the putative 20-HETE antagonist, 20-hydroxyeicosa-5(Z),14(Z)-dienoic acid. In vivo, implantation of U251O cells in the brain of nude rats resulted in a ϳ10-fold larger tumor volume (10 days postimplantation) compared with animals receiving mock-transfected U251 cells. These data show that elevations in 20-HETE synthesis in U251 cells lead to an increased growth both in vitro and in vivo. This suggests that 20-HETE may have proto-oncogenic properties in U251 human gliomas. Further studies are needed to determine whether 20-HETE plays a role promoting growth of some human gliomas.