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: 31.48). 04/2008; 319(5871):1834-7. DOI: 10.1126/science.1153264
Source: PubMed

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.

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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 · 7.21 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). "
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    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. DOI:10.1016/j.antiviral.2014.07.002 · 3.94 Impact Factor
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    • "However, the exact function of the flavivirus NS3hel in the virus replication cycle is unknown. It is speculated that NS3hel could resolve secondary structures of the genomic RNA, displace transacting protein cofactors, and/or separate the dsRNA intermediate that is transiently formed during the polymerization reaction catalyzed by NS5 RdRP into single-strand form (Malet H, et al., 2007; Yu I M, et al., 2008; Yu L, et al., 2008). "
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    ABSTRACT: Flaviviruses are positive-sense RNA viruses, and many are important human pathogens. Nonstructural protein 2B and 3 of the flaviviruses (NS2BNS3) form an endoplasmic reticulum (ER) membraneassociated hetero-dimeric complex through the NS2B transmembrane region. The NS2BNS3 complex is multifunctional. The N-terminal region of NS3, and its cofactor NS2B fold into a protease that is responsible for viral polyprotein processing, and the C-terminal domain of NS3 possesses NTPase/RNA helicase activities and is involved in viral RNA replication and virus particle formation. In addition, NS2BNS3 complex has also been shown to modulate viral pathogenesis and the host immune response. Because of the essential functions that the NS2BNS3 complex plays in the flavivirus life cycle, it is an attractive target for antiviral development. This review focuses on the recent biochemical and structural advances of NS2BNS3 and provides a brief update on the current status of drug development targeting this viral protein complex.
    Virologica Sinica 03/2014; DOI:10.1007/s12250-014-3438-6
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