The recent rapid spread of Zika virus and its unexpected linkage to birth defects and an autoimmune-neurological syndrome has generated worldwide concern. Zika virus is a flavivirus like dengue, yellow fever and West Nile viruses. We present the 3.8Å resolution structure of mature Zika virus determined by cryo-electron microscopy. The structure of Zika virus is similar to other known flavivirus structures except for the ~10 amino acids that surround the Asn154 glycosylation site found in each of the 180 envelope glycoproteins that make up the icosahedral shell. The carbohydrate moiety associated with this residue, recognizable in the cryo-EM electron density, may function as an attachment site of the virus to host cells. This region varies not only among Zika virus strains but also in other flaviviruses and suggests that changes in this region influence virus transmission and disease.

Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus genus, which is a member of the Flaviviridae family. They are human pathogens that cause large epidemics and tens of thousands of deaths annually in many parts of the world. The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion. This review focuses mainly on structural studies of dengue virus.

The first structure of a flavivirus has been determined by using a combination of cryoelectron microscopy and fitting of the known structure of glycoprotein E into the electron density map. The virus core, within a lipid bilayer, has a less-ordered structure than the external, icosahedral scaffold of 90 glycoprotein E dimers. The three E monomers per icosahedral asymmetric unit do not have quasiequivalent symmetric environments. Difference maps indicate the location of the small membrane protein M relative to the overlaying scaffold of E dimers. The structure suggests that flaviviruses, and by analogy also alphaviruses, employ a fusion mechanism in which the distal beta barrels of domain II of the glycoprotein E are inserted into the cellular membrane.

Dengue virus (DENV) modifies cellular membranes to establish its sites of replication. Although the 3D architecture of these structures has recently been described, little is known about the cellular pathways required for their formation and expansion. In this report, we examine the host requirements for DENV replication using a focused RNAi analysis combined with validation studies using pharmacological inhibitors. This approach identified three cellular pathways required for DENV replication: autophagy, actin polymerization, and fatty acid biosynthesis. Further characterization of the viral modulation of fatty acid biosynthesis revealed that a key enzyme in this pathway, fatty acid synthase (FASN), is relocalized to sites of DENV replication. DENV nonstructural protein 3 (NS3) is responsible for FASN recruitment, inasmuch as (i) NS3 expressed in the absence of other viral proteins colocalizes with FASN and (ii) NS3 interacts with FASN in a two-hybrid assay. There is an associated increase in the rate of fatty acid biosynthesis in DENV-infected cells, and de novo synthesized lipids preferentially cofractionate with DENV RNA. Finally, purified recombinant NS3 stimulates the activity of FASN in vitro. Taken together, these experiments suggest that DENV co-opts the fatty acid biosynthetic pathway to establish its replication complexes. This study provides mechanistic insight into DENV membrane remodeling and highlights the potential for the development of therapeutics that inhibit DENV replication by targeting the fatty acid biosynthetic pathway.is the causative agent of dengue fever, dengue hemorrhagic fever, and toxic shock syndrome (1). These diseases are prevalent in tropical regions around the world, where the mosquito vectors thrive. A total of 50 to 100 million DENV-related infections occur annually worldwide (2). Despite the large burden to human health, basic research into the development of DENV antiviral therapy has been limited.All positive-strand RNA viruses remodel cytosolic membranes to establish sites of replication (reviewed in ref.3). These structures play a critical role in the viral life cycle, likely by increasing the local concentration of replicating viral components and by sequestering viral antigens from recognition by host immune surveillance mechanisms. Following the initial translation and processing of DENV proteins, cellular membranes are remodeled to establish cytosolic replication complexes (RCs). DENV nonstructural protein 4A (NS4A) has been proposed to be sufficient for DENV membrane remodeling, perhaps performing a structural role by inducing membrane curvature (4). This function requires a processing event in which the C terminus of NS4A is removed by the viral NS2B-3 protease.Recently, the 3D structure of DENV RCs has been determined by electron tomography (5). That study clearly demonstrated that viral replication takes place on double-membrane vesicles that are contiguous with the endoplasmic reticulum (ER). Interestingly, there also appears to be physical linkage between sit...

Flaviviruses, the human pathogens responsible for dengue fever, West Nile fever, tick-borne encephalitis and yellow fever, are endemic in tropical and temperate parts of the world. The flavivirus non-structural protein 1 (NS1) functions in genome replication as an intracellular dimer and in immune system evasion as a secreted hexamer. We report crystal structures for full-length, glycosylated NS1 from West Nile and dengue viruses. The NS1 hexamer in crystal structures is similar to a solution hexamer visualized by single-particle electron microscopy. Recombinant NS1 binds to lipid bilayers and remodels large liposomes into lipoprotein nanoparticles. The NS1 structures reveal distinct domains for membrane association of the dimer and interactions with the immune system, and are a basis for elucidating the molecular mechanism of NS1 function.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

Intracellular cleavage of immature flaviviruses is a critical step in assembly that generates the membrane fusion potential of the E glycoprotein. With cryo-electron microscopy we show that the immature dengue particles undergo a reversible conformational change at low pH that renders them accessible to furin cleavage. At a pH of 6.0, the E proteins are arranged in a herringbone pattern with the pr peptides docked onto the fusion loops, a configuration similar to that of the mature virion. After cleavage, the dissociation of pr is pH-dependent, suggesting that in the acidic environment of the trans-Golgi network pr is retained on the virion to prevent membrane fusion. These results suggest a mechanism by which flaviviruses are processed and stabilized in the host cell secretory pathway.

Dengue virus is responsible for Ϸ50 -100 million infections, resulting in nearly 24,000 deaths annually. The capsid (C) protein of dengue virus is essential for specific encapsidation of the RNA genome, but little structural information on the C protein is available. We report the solution structure of the 200-residue homodimer of dengue 2 C protein. The structure provides, to our knowledge, the first 3D picture of a flavivirus C protein and identifies a fold that includes a large dimerization surface contributed by two pairs of helices, one of which has characteristics of a coiled-coil. NMR structure determination involved a secondary structure sorting approach to facilitate assignment of the intersubunit nuclear Overhauser effect interactions. The dimer of dengue C protein has an unusually high net charge, and the structure reveals an asymmetric distribution of basic residues over the surface of the protein. Nearly half of the basic residues lie along one face of the dimer. In contrast, the conserved hydrophobic region forms an extensive apolar surface at a dimer interface on the opposite side of the molecule. We propose a model for the interaction of dengue C protein with RNA and the viral membrane that is based on the asymmetric charge distribution of the protein and is consistent with previously reported results.D engue virus, a member of the flavivirus genus of enveloped RNA viruses, is one of the most significant mosquito-borne viral pathogens, given the impact of the recent resurgence of dengue fever and dengue hemorrhagic fever (1). Other members of the flavivirus genus are also important human pathogens and include such viruses as yellow fever, West Nile virus, and Japanese encephalitis (2). The structure of dengue virus was recently described by using cryo-electron microscopy, which permitted visualization of certain viral protein components (3). The organization of the major envelope glycoprotein (E) in the virion was obtained by fitting the atomic structure of the ectodomain from the related tick-borne encephalitis (TBE) E protein into the outermost layer of density (4). Density internal to the E ectodomain demonstrated a host-derived lipid bilayer with transmembrane helices from E and the small structural protein M (5). Interior to the bilayer is the nucleocapsid core that comprises multiple copies of the capsid, or core, protein (C) and a single 10.7-kb genome RNA. Surprisingly, the organization of the C protein within the nucleocapsid core layer is not discernable. The lack of strong density for C may suggest a rather unique architecture of the flavivirus nucleocapsid core that is distinct from the structural organization of morphologically related viruses such as alphaviruses, a genus in the Togaviridae family of mosquito-borne viruses.The dengue C protein is essential in virus assembly to ensure specific encapsidation of the viral genome. The critical role of C is evident from the existence of subviral particles that are released from infected cells but lack C protein and genome RNA (6). The me...