Structure of the Immature Dengue Virus at Low pH Primes Proteolytic Maturation

Department of Biological Sciences, 915 West State Street, Purdue University, West Lafayette, IN 47907-2054, USA.
Science (Impact Factor: 33.61). 04/2008; 319(5871):1834-7. DOI: 10.1126/science.1153264
Source: PubMed


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.

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    • "prM can be cleaved into a C-terminal M portion, which remains associated with the virus particle. The other is an N-terminal 91-amino acid precursor (pr) peptide that dissociates upon release from the virion (Yu et al. 2008) by the cellular endoprotease furin, leading to the formation of mature infectious virions. The cleavage of prM protein is required for the activation of DENV infectivity. "
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    ABSTRACT: Severe dengue is more likely found during secondary heterologous dengue virus (DENV) infection or primary infection of infants born to dengue-immune mothers and led to the hypothesis of antibody-dependent enhancement (ADE). It has been reported that pre-membrane (prM)-reactive antibodies do not efficiently neutralize DENV infection but instead potently promote ADE infection. Meanwhile, these enhancing anti-prM antibodies mainly react with the precursor (pr) peptide. To evaluate the effect of pr gene substitution on neutralization and ADE of DENV infection, a novel chimeric dengue virus (JEVpr/DENV2) was rationally constructed by replacing the DENV pr gene with Japanese encephalitis virus (JEV) pr gene, based on the full-length infectious complementary DNA (cDNA) clone of DENV2 ZS01/01. We found that chimeric JEVpr/DENV2 showed reduced virulence and good immunogenicity. In addition, anti-JEVpr/DENV2 sera showed broad cross-reactivity and efficient neutralizing activity with all four DENV serotypes and immature DENV2 (ImDENV2). Most importantly, compared with anti-DENV2 sera, anti-JEVpr/DENV2 sera showed significantly reduced enhancing activity of DENV infection in K562 cells. These results suggest that the ADE activities could be reduced by replacing the DENV pr gene with JEV pr gene. These findings may help us better understand the pathogenesis of DENV infection and provide a reference for the development of a vaccine against DENV.
    Applied Microbiology and Biotechnology 07/2015; 99(22). DOI:10.1007/s00253-015-6819-3 · 3.34 Impact Factor
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    • "It has been reported that DENV glycoprotein prME undergoes a conformational change in the Golgi apparatus, possibly caused by luminal acidification, which leads to the formation of E homodimers (Li et al., 2008; Yu et al., 2008). RSP released by F&T showed that the percentage of E/E homodimers was 3-to 4-fold higher in cells expressing wild-type prME in comparison to Triple prME, which behaved in similar fashion to R6S (Figure 6F), corroborating immunofluorescence observations (Figures 6B–6D). "
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    ABSTRACT: Membrane receptors at the surface of target cells are key host factors for virion entry; however, it is unknown whether trafficking and secretion of progeny virus requires host intracellular receptors. In this study, we demonstrate that dengue virus (DENV) interacts with KDEL receptors (KDELR), which cycle between the ER and Golgi apparatus, for vesicular transport from ER to Golgi. Depletion of KDELR by siRNA reduced egress of both DENV progeny and recombinant subviral particles (RSPs). Coimmunoprecipitation of KDELR with dengue structural protein prM required three positively charged residues at the N terminus, whose mutation disrupted protein interaction and inhibited RSP transport from the ER to the Golgi. Finally, siRNA depletion of class II Arfs, which results in KDELR accumulation in the Golgi, phenocopied results obtained with mutagenized prME and KDELR knockdown. Our results have uncovered a function for KDELR as an internal receptor involved in DENV trafficking. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 03/2015; 10(9). DOI:10.1016/j.celrep.2015.02.021 · 8.36 Impact Factor
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    • "Next the Fig. 1. Possible hemifusion route to virus (upper)–host (lower) membrane fusion, illustrated through alignment of E protein to: a) dimeric, mature viral assembly (3C6R [101]); b) an intermediate structure during trimerization approximated by the two cryo-EM structures with exposed fusion loops, 3C6D [101] and 3IXY [9]; c) target, fused state with trimeric form (1OK8 [6]) as proposed in earlier works [6] [77], arbitrarily positioned to interact with a catenoid-shaped, zero mean curvature, membrane. Panels (b) and (d) are marked by * to illustrate the state defining the free energy barrier for this process. "
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    ABSTRACT: Dengue virus is coated by an icosahedral shell of 90 envelope protein dimers that convert to trimers at low pH and promote fusion of its membrane with the membrane of the host endosome. We provide the first estimates for the free energy barrier and minimum for two key steps in this process: host membrane bending and protein-membrane binding. Both are studied using complementary membrane elastic, continuum electrostatics and all-atom molecular dynamics simulations. The predicted host membrane bending required to form an initial fusion stalk presents a 22-30 kcal/mol free energy barrier according to a constrained membrane elastic model. Combined continuum and molecular dynamics results predict a 15 kcal/mol free energy decrease on binding of each trimer of Dengue envelope protein to a membrane with 30% anionic phosphatidylglycerol lipid. The bending cost depends on the preferred curvature of the lipids composing the host membrane leaflets, while the free energy gained for protein binding depends on the surface charge density of the host membrane. The fusion loop of the envelope protein inserts exactly at the level of the interface between the membrane's hydrophobic and head-group regions. The methods used in this work provide a means for further characterization of the structures and free energies of protein-assisted membrane fusion. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.
    Biochimica et Biophysica Acta (BBA) - Biomembranes 01/2015; 1848(4). DOI:10.1016/j.bbamem.2014.12.018 · 3.84 Impact Factor
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