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.

Chikungunya virus (CHIKV) has infected millions of people in Africa, Europe, and Asia1,2 since its re-emergence in Kenya in 2004. The severity of disease and spread of this epidemic virus present a serious public health threat in the absence of vaccines or anti-viral therapies. Here, we describe a novel vaccine that protects against emerging CHIKV infection of non-human primates (NHP). We show that selective expression of viral structural proteins gives rise to virus-like particles (VLPs) in vitro that resemble replication-competent alphaviruses. Immunization with these VLPs elicited neutralizing antibodies against envelope proteins from different CHIKV strains. Monkeys immunized with VLPs produced high titer neutralizing antibodies that protected against viremia after high dose challenge. We transferred these antibodies into immunodeficient mice, where they protected against subsequent lethal CHIKV challenge, establishing a humoral mechanism of protection. Immunization with alphavirus VLP vaccines represents a strategy to contain the spread of CHIKV and related pathogenic viruses in humans.

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.

A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment.DOI: http://dx.doi.org/10.7554/eLife.00435.001

Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.Maturation, by which a noninfectious immature virus particle is converted to an infectious virion, is an essential step in the replication cycle of many viruses. It often involves the proteolytic processing of a precursor protein coupled with the conformational transformation of the virion. The assembly pathway for flaviviruses is well established, and the structures of the entire virion as well as individual glycoproteins representing different stages of the life cycle have been determined. Thus, flaviviruses are an excellent system for investigating the dynamic aspects of maturation in enveloped viruses.Flavivirus maturation requires furin (21), a cellular protease located primarily in the trans-Golgi network (TGN) (16). Immature particles, containing heterodimers of the precursor membrane protein (prM) and the envelope protein (E), bud into the endoplasmic reticulum (ER) and then are transported through the cellular secretory pathway to the extracellular environment (12, 23). The cleavage of prM by furin generates the membrane-anchored protein M and the soluble product pr. In the mature, infectious virion, the pr peptide is absent, and the virus undergoes membrane fusion in the endosome at low pH. In contrast, immature particles produced from furin-deficient LoVo cells, or cells grown in the presence of protease inhibitors or acidotropic reagents to prevent furin cleavage, contain prM and are significantly less infectious (21).Crystal structures of the E protein (9,14,18,19,29) show that each polypeptide chain contains three domains: the structurally central amino-terminal domain (DI), the dimerization domain (DII) containing the fusion loop, and the carboxyterminal immunoglobulin-like domain (DIII). In the presence of lipids, low pH induces rearrangements of the E proteins, resulting in the formation of homotrimers with the fusion loops and the C-terminal membrane anchors located at the same end (4, 15). The conformational change of the E proteins presumably facilitates the merging of the viral membrane with the endosomal membrane, the...

During dengue virus replication, an incomplete cleavage of the envelope glycoprotein prM, generates a mixture of mature (prM-less) and prM-containing, immature extracellular particles. In this study, sequential immunoprecipitation and cryoelectron microscopy revealed a third type of extracellular particles, the partially mature particles, as the major prM-containing particles in a dengue serotype 2 virus. Changes in the proportion of viral particles in the pr-M junction mutants exhibiting altered levels of prM cleavage suggest that the partially mature particles may represent an intermediate subpopulation in the virus maturation pathway. These findings are consistent with a model suggesting the progressive mode of prM cleavage.Dengue viruses are enveloped, positive-strand RNA viruses in the genus Flavivirus of the family Flaviviridae (19). The viral genome encodes three structural proteins (C, prM/M, and E) and seven nonstructural proteins (19). Two types of genomecontaining particles, the immature and mature particles, can be distinguished by the differences in size and surface morphology and the presence and cleavage status of the envelope glycoprotein prM (19,20). The immature particles are assembled in the endoplasmic reticulum as spherical "spiky" particles of about 60 nm in diameter (36). Each of the spikes is formed by a noncovalent association of three prM-E heterodimers, with the pr portion of prM on the outermost part of the spike providing the main contact (18,36). During the export, the low-pH environment of the trans-Golgi network induces the rearrangement of prM-E heterodimers into a flattened conformation that allows for an internal cleavage of prM by furin (34). The complete prM cleavage generates the mature particles, which are about 50 nm in diameter and present a smooth surface (17). These infectious particles contain 90 E homodimers arranged in groups of three parallel dimers in the "herringbone" pattern (17). Further complexity of the viral particles was observed in studies of dengue virus and West Nile virus in the form of particles having an intermediate conformation between those of the mature and immature particles (3,24,35).Cleavage of prM is a prerequisite for an acquisition of infectivity, as the pr portion of prM functions as a mechanical barrier to protect the fusion loop in the receptor-binding E glycoprotein from undergoing low pH-mediated fusion (7,18,29). Inhibition of the prM cleavage by mutation of the furin cleavage site, treatment of the infected cells with acidotropic amines, or growth of the virus in furin-deficient cells generates noninfectious particles in the extracellular compartment (7,8,10,25,37). During the replication of dengue virus, cleavage of prM is, however, usually incomplete (1,4,9,11,16,21,25,27,32,33). This reflects an inhibition of cleavage mediated by a highly conserved acidic residue at the P3 cleavage position of the pr-M junction (15). Currently, it is not clear how the prM molecules are collectively cleaved in each viral particle. In the "all-or-none...

Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.antibody | cryoelectron microscopy | flavivirus W est Nile virus (WNV) is a human pathogen that causes a febrile illness, which can progress to encephalitis, paralysis, and death. The virus is endemic in parts of Africa, Asia, and Europe, and in the past decade has spread throughout North America and into Central and South America (1). WNV is closely related to other arthropod-transmitted, medically relevant flaviviruses, such as dengue, yellow fever, Japanese encephalitis, and tick-borne encephalitis viruses. These lipid-enveloped viruses enter host cells by receptor-mediated endocytosis. The singlestranded, positive-sense RNA genome is released into the cytoplasm after low pH induces the fusion of the viral lipid envelope with the endosomal membrane.Mature WNV virions are roughly spherical with a diameter of about 500 Å. The outer viral surface is composed of an icosahedral scaffold of 180 closely packed copies of the membrane-anchored envelope (E) glycoprotein. Sets of three, nearly parallel E homodimers are associated into rafts that form a herringbone pattern on the surface of mature virions. The ectodomain of E has three structural domains, DI, DII, and DIII (2-5), with domain DI positioned structurally between DII and DIII. DII contains a fusion loop at its distal end that is indispensable for virus-cell membrane fusion. The Ig-like C-terminal domain DIII undergoes a major, pH-triggered positional rearrangement essential for fusion, and may also be involved in receptor binding (2, 6-12). During cell entry of flaviviruses, low endosomal pH triggers a proposed E protein rearrangement cascade, including the dissociation of E dimers and the outward rotation of DII during the repositioning of E monomers into fusion-active trimers (6, 9, 13).The humoral immune response is essential for protection against flavivirus infection and disease (14,15). The E glycoprotein is the principal antigen that elicits neutralizing antibodies agai...

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