Structure of the immature dengue virus at low pH primes proteolytic maturation
ABSTRACT 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.
- SourceAvailable from: Thibault Lagache
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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). "
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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- "Virus maturation occurs in the TGN, where a cellular protease, probably furin, cleaves prM, generating M protein (8 kDa) and the 'pr' peptide (Zhang et al., 2003a). The TGN acidic environment induces conformational changes in the prM:E heterodimer that expose the cleavage site (Randolph, Winkler and Stollar 1990; Yu et al., 2008). prM interacts and stabilizes E protein DII, as the pr sequence β-barrel structure shields the E protein fusion loop, preventing conformational changes that could activate its fusogenic activity during TGN secretory pathway (Zhang et al., 2003a; Li et al., 2008; Yu et al., 2008; Zheng, Umashankar and Kielian 2010). "
ABSTRACT: Dengue is the most prevalent arthropod-borne viral disease, caused by dengue virus, a member of the Flaviviridae family. Its worldwide incidence is now a major health problem, with 2.5 billion people living in risk areas. In this review, we integrate the structural rearrangements of each viral protein and their functions in all the steps of virus entry into the host cells. We describe in detail the putative receptors and attachment factors in mammalian and mosquito cells, and the recognition of viral immunocomplexes via Fcγ receptor in immune cells. We also discuss that virus internalization might occur through distinct entry pathways, including clathrin-mediated or non-classical clathrin-independent endocytosis, depending on the host cell and virus serotype or strain. The implications of viral maturation in virus entry are also explored. Finally, we discuss the mechanisms of viral genome access to the cytoplasm. This includes the role of low pH-induced conformational changes in the envelope protein that mediate membrane fusion, and original insights raised by our recent work that supports the hypothesis that capsid protein would also be an active player in this process, acting on viral genome translocation into the cytoplasm. © FEMS 2014. All rights reserved. For permissions, please e-mail: email@example.com.FEMS Microbiology Reviews 12/2014; 39(2). DOI:10.1093/femsre/fuu004 · 13.81 Impact Factor
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- "The virion is a spherical particle of approximately 50 nm in diameter and contains a lipid envelope harboring two structural proteins, the envelope (E) and membrane proteins (prM in immature virions and M in mature virions). The envelope surrounds the nucleocapsid, containing the capsid protein (C) associated with the single stranded RNA genome of positive polarity (Kuhn et al., 2002; Mukhopadhyay et al., 2005; Yu et al., 2008). The first step in DENV infection is the viral binding to the cellular receptor molecules on the surface of the target cell, followed by receptor-mediated endocytosis and subsequent fusion of the viral and endosomal membranes leading to genome release into the cytoplasm (Hidari and Suzuki, 2011). "
ABSTRACT: Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti- inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 hours with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly.Antiviral Research 07/2014; 109(1). DOI:10.1016/j.antiviral.2014.07.002 · 3.94 Impact Factor